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STRIPPING VOLTAMMETRIC METHODS FOR THE DETERMINATION OF AFLATOXIN COMPOUNDS

MOHAMAD HADZRI BIN YAACOB

UNIVERSITI TEKNOLOGI MALAYSIA

BAHAGIAN A – Pengesahan Kerjasama * Adalah disahkan bahawa projek penyelidikan tesis ini telah dilaksanakan melalui kerjasama antara _____________________ dengan _________________________ Disahkan oleh: Tandatangan

: ..........................................................

Nama

: ..........................................................

Tarikh : ..........................

Jawatan:........................................................... (Cop rasmi) * Jika penyediaan tesis/projek melibatkan kerjasama. BAHAGIAN B – Untuk Kegunaan Pejabat Sekolah Pengajian Siswazah Tesis ini telah diperiksa dan diakui oleh: Nama dan Alamat Pemeriksa Luar

:

Prof. Dr. Noor Azhar Bin Mohd Shazili Pengarah Institut Oseanografi, Kolej Universiti Sains dan Teknologi Malaysia, Mengabang Telipot 21030 Kuala Terengganu

Nama dan Alamat Pemeriksa Dalam I

:

Prof. Madya Dr. Razali Bin Ismail Fakulti Sains, UTM, Skudai

Pemeriksa Dalam II

:

Nama Penyelia Lain (jika ada)

:

Disahkan oleh Penolong Pendaftar di Sekolah Pengajian Siswazah: Tandatangan Nama

: .......................................................... : .GANESAN A/L ANDIMUTHU

Tarikh : ..........................

STRIPPING VOLTAMMETRIC METHODS FOR THE DETERMINATION OF AFLATOXIN COMPOUNDS

MOHAMAD HADZRI BIN YAACOB

A thesis submitted in fulfilment of the requirements for the award of the degree of Doctor of Philosophy

Faculty of Science Universiti Teknologi Malaysia

APRIL 2006

ii

iii

Specially dedicated to:

My mother, wife, sons, daughters and all families for all the love, support and continuous prayer for my success in completing this work.

iv

ACKNOWLEDGEMENT All praise be to ALLAH SWT and blessing be upon His Prophet SAW whose ultimate guidance creates a more meaningful purpose to this work. I wish to express my sincere gratitude and appreciation to the people who have both directly and indirectly contributed to this thesis. The following are those to whom I am particularly indebted: My supervisors A.P. Dr. Abdull Rahim bin Hj. Mohd. Yusoff and Prof. Dr. Rahmalan Ahamad for their invaluable guidance, freedom of work and constant encouragement throughout the course of this work. School Of Health Sciences, USM, Health Campus, Kubang Krian Kelantan for awarding study leave together with scholarship in completing the work. Prof Baharuddin Saad from USM Penang, AP Dr. Razali Ismail from Chemistry Department, Faculty of Science, UTM and Prof Barek from Charles University, Prague, Czeck Republic for their useful discussion and suggestion. Also to Mr Radwan Ismail from Department of Chemistry, Penang Branch, Mrs Marpongahtun Misni and Mr Wan Kamaruzaman Wan Ahmad for their friendship, ideas and continuous support in carrying out this work. Also to Mr Mat Yasin bin Sirin, Mrs Ramlah binti Husin and Mr Azmi Mahmud for their assistance throughout the work. UTM for awarding Short Term Grant No: 75152 / 2004 My mother, wife and all families for their encouragements, supports, patience, tolerance and understanding.

v

ABSTRACT

Aflatoxin, which is produced by Aspergillus flavus and Aspergillus parasiticus fungi is one of the compounds in the mycotoxin group. The main types of aflatoxins are AFB1, AFB2, AFG1 and AFG2 which have carcinogenic properties and are dangerous to human health. Various techniques have been used for their measurements such as the high performance liquid chromatography (HPLC), enzyme linked immunosorbant assay (ELISA) and radioimmunoassay (RIA) but all these methods have disadvantages such as long analysis time, consume a lot of reagents and expensive. To overcome these problems, the voltammetric technique was proposed in this study using controlled growth mercury drop (CGME) as the working electrode and Britton Robinson buffer (BRB) as the supporting electrolyte. The voltammetric methods were used for investigating the electrochemical properties and the quantitative analysis of aflatoxins at the mercury electrode. The experimental conditions were optimised to obtain the best characterised peak in terms of peak height with analytical validation of the methods for each aflatoxin. The proposed methods were applied for the analysis of aflatoxins in groundnut samples and the results were compared with those obtained by the HPLC technique. All aflatoxins were found to adsorb and undergo irreversible reduction reaction at the working mercury electrode. The optimum experimental parameters for the differential pulse cathodic stripping voltammetry (DPCSV) method were the BRB at pH 9.0 as the supporting electrolyte, initial potential (Ei): -0.1 V, final potential (Ef): -1.4 V, accumulation potential (Eacc): 0.6 V, accumulation time (tacc): 80 s, scan rate: 50 mV/s and pulse amplitude: 80 mV. The optimum parameters for the square wave stripping voltammetry (SWSV) method were Ei = -0.1 V, Ef = -1.4 V, Eacc: -0.8 V, tacc: 100 s, scan rate: 3750 mV/s, frequency: 125 Hz and voltage step: 30 V. At the concentration of 0.10 µM, using DPCSV method with the optimum parameters, AFB1, AFB2, AFG1 and AFG2 produced a single peak at -1.21 V, -1.23 V, -1.17 V and -1.15 V (versus Ag/AgCl) respectively. Using the SWSV method, a single peak appeared at -1.30 V for AFB1 and AFB2 while -1.22 V for AFG1 and AFG2. The calibration curves for all aflatoxins were linear with the limit of detection (LOD) of approximately 2.0 ppb and 0.50 ppb obtained by the DPCSV and SWSV methods respectively. The results of aflatoxins content in individual groundnut samples do not vary significantly when compared with those obtained by the HPLC technique. Finally, it can be concluded that both proposed methods which are accurate, precise, robust, rugged, fast and low cost were successfully developed and are potential alternative methods for routine analysis of aflatoxins in groundnut samples.

vi

ABSTRAK

Aflatoksin adalah sejenis sebatian yang dihasilkan oleh kulat Aspergillus flavus dan Aspergillus parasiticus yang digolongkan di dalam kumpulan mikotoksin. Jenis utama aflatoksin adalah AFB1, AFB2, AFG1 dan AFG2 yang bersifat karsinogen serta merbahaya kepada kesihatan manusia. Pelbagai teknik telah digunakan untuk menentukan aflatoksin seperti kromatografi cecair prestasi tinggi (HPLC), asai serapan imuno berikatan enzim (ELISA) dan radioamunoasai (RIA) tetapi teknik-teknik ini mempunyai kelemahan seperti masa analisis yang panjang, melibatkan reagen yang banyak dan kos yang mahal. Untuk mengatasi masalah ini, teknik voltammetri telah dicadangkan untuk kajian aflatoksin menggunakan titisan raksa pembesaran terkawal (CGME) sebagai elektrod bekerja dan larutan penimbal Britton-Robinson (BRB) sebagai elektrolit penyokong. Pelbagai kaedah voltammetri telah digunakan untuk mengkaji sifat elektrokimia aflatoksin pada elektrod raksa dan analisis kuantitatifnya. Parameter kajian telah dioptimumkan untuk memperolehi puncak yang elok berdasarkan ketinggian puncak serta pengesahan analisis untuk kaedah yang dibangunkan bagi setiap aflatoksin. Kaedah ini telah digunakan untuk menentukan kandungan aflatoksin di dalam sampel kacang tanah di mana keputusan yang diperolehi telah dibandingkan dengan keputusan HPLC. Semua aflatoksin yang dikaji didapati terjerap dan menjalani proses tindakbalas penurunan tidak berbalik pada elektrod raksa. Parameter optimum untuk kaedah voltammetri perlucutan kathodik denyut pembeza (DPCSV) adalah larutan BRB pada pH 9.0 sebagai larutan elektrolit, keupayaan awal (Ei): -1.0 V, keupayaan akhir (Ef): -1.4 V, keupayaan pengumpulan (Eacc): -0.6 V, masa pengumpulan (tacc): 80 s, kadar imbasan: 50 mV/s dan amplitud denyut: 80 mV. Untuk kaedah voltammetri perlucutan gelombang bersegi (SWSV), parameter optimum adalah Ei : -1.0 V, Ef : -1.4 V, Eacc: -0.8 V, tacc: 100 s, kadar imbasan: 3750 mV/s, frekuensi: 125 Hz dan beza keupayaan: 30 mV. Menggunakan parameter optimum untuk DPCSV, 0.10 µM AFB1, AFB2, AFG1 dan AFG2 menghasilkan puncak tunggal pada keupayaan -1.21 V, -1.23 V, -1.17 V dan -1.15 V (melawan Ag/AgCl) masingmasingnya. Menggunakan kaedah SWSV, puncak terhasil pada -1.30 V untuk AFB1 dan AFB2, -1.22 V untuk AFG1 dan AFG2. Keluk kalibrasi adalah linear untuk semua aflatoksin dengan had pengesanan (LOD) pada 2.0 dan 0.5 ppb diperolehi dari kaedah DPCSV dan SWSV masing-masingnya. Keputusan analisis kandungan aflatoksin di dalam sampel kacang tanah tidak memberi perbezaan ketara berbanding dengan yang diperolehi menggunakan teknik HPLC. Kesimpulannya, kedua-dua kaedah yang dikaji yang merupakan kaedah yang tepat, jitu, cepat, sesuai digunakan dengan pelbagai model voltammetri dan kos yang murah telah berjaya dibangunkan dan berpotensi besar menjadi kaedah alternatif untuk analisis kandungan aflatoksin di dalam kacang tanah secara berkala.

vii

TABLE OF CONTENTS

CHAPTER

1

TITLE

PAGE

TITLE

i

DECLARATION

ii

DEDICATION

iii

ACKNOWLEDGEMENT

iv

ABSTRACT

v

ABSTRAK

vi

TABLE OF CONTENTS

vii

LIST OF TABLES

xiii

LIST OF FIGURES

xvi

ABBREVATIONS

xxix

LIST OF APPENDICES

xxxiii

LITERATURE REVIEW

1

1.1

Overview

1

1.2

Aflatoxins

3

1.2.1 Aflatoxins in general

3

1.2.2

Chemistry of aflatoxins

5

1.2.3

Health aspects of aflatoxins

12

1.2.4

Analytical methods for the determination of

16

aflatoxins 1.2.5 1.3.

Electrochemical properties of aflatoxins

28

Voltammetric technique

30

1.3.1

30

Voltammetric techniques in general

viii 1.3.2

Voltammetric measurement

31

1.3.2.1 Instrumentation

31

1.3.2.2 Solvent and supporting electrolyte

44

1.3.2.3 Current in voltammetry

46

1.3.2.4 Quantitative and quantitative aspects of

48

voltammetry 1.3.3

Type of voltammetric techniques

49

1.3.3.1 Polarography

49

1.3.3.2 Cyclic voltammetry

51

1.3.3.3 Stripping voltammetry

54

1.3.3.3a Anodic stripping voltammetry

56

1.3.3.3b Cathodic stripping voltammetry

57

1.3.3.3c Adsorptive stripping voltammetry 58 1.3.3.4 Pulse voltammetry

1.4

2

59

1.3.3.4a Differential pulse voltammetry

60

1.3.3.4b Square wave voltammetry

61

Objective and scope of study

64

1.4.1

Objective of study

64

1.4.2

Scope of study

67

RESEARCH METHODOLOGY

70

2.1

Apparatus, material and reagents

70

2.1.1

Apparatus

70

2.1.2

Materials

72

2.1.2.1 Aflatoxin stock and standard solutions

72

2.1.2.2 Real samples

73

Reagents

73

2.1.3.1 Britton Robinson buffer, 0.04 M

73

2.1.3.2 Carbonate buffer, 0.04 M

74

2.1.3.3 Phosphate buffer, 0.04 M

74

2.1.3

ix 2.1.3.4 Ascorbic acid

74

2.1.3.5 β-cyclodextrin solution, 1.0 mM -5

75

2.1.3.6 L-Cysteine, 1.0 x 10 M

75

2.1.3.7 2,4-dihydrofuran, 0.15 M

75

-2

2.1.3.8 Coumarin, 3.0 x 10 M

75

2.1.3.9 Poly-L-lysine, 10 ppm

75

2.1.3.10 Standard aluminium (II) solution, 1.0 mM 75

2.2

2.1.3.11 Standard plumbum(II) solution, 1.0 mM

76

2.1.3.12 Standard zinc (II) solution, 1.0 mM

76

2.1.3.13 Standard copper (II) solution, 1.0 mM

76

2.1.3.14 Standard nickel (II) solution, 1.0 mM

76

2.1.3.15 Methanol: 0.1 N HCl solution, 95%

76

2.1.3.16 Zinc sulphate solution, 15%

76

Analytical Technique

77

2.2.1

General procedure for voltammetric analysis

77

2.2.2

Cyclic voltammetry (Anodic and cathodic

77

directions)

2.2.3

2.2.2.1 Standard addition of sample

77

2.2.2.2 Repetitive cyclic voltammetry

78

2.2.2.3 Effect of scan rate

78

Differential pulse cathodic stripping

78

voltammetric determination of AFB2 2.2.3.1 Effect of pH

79

2.2.3.2 Method optimisation for the determination 79 of AFB2 2.2.3.2a Effect of scan rate

79

2.2.3.2b Effect of accumulation potential

80

2.2.3.2c Effect of accumulation time

80

2.2.3.2d Effect of initial potential

80

2.2.3.2e Effect of pulse amplitude

80

x 2.2.3.3 Method validation

80

2.2.3.4 Interference studies

81

2.2.3.4a Effect of Cu(II), Ni (II), Al(III),

81

Pb(II) and Zn(II) 2.2.3.4b Effect of ascorbic acid,

82

β-cyclodextrin and L-cysteine 2.2.4

2.2.3.5 Modified mercury electrode with PLL

82

Square wave cathodic stripping voltammetry

82

(SWSV)

2.2.5

2.2.4.1 SWSV parameters optimisation

82

2.2.4.2 SWSV determination of all aflatoxins

82

Stability studies of aflatoxins

83

2.2.5.1 Stability of 10 ppm aflatoxins

83

2.2.5.2 Stability of 1 ppm aflatoxins

83

2.2.5.3 Stability of 0.1 µM aflatoxins exposed

83

to ambient temperature 2.2.5.4 Stability of 0.1 µM aflatoxins in different

84

pH of BRB

3

2.2.6 Application to food samples

84

2.2.6.1 Technique 1

84

2.2.6.2 Technique 2

84

2.2.6.3 Technique 3

85

2.2.6.4 Blank measurement

85

2.2.6.5 Recovery studies

85

2.2.6.6 Voltammetric analysis

86

RESULTS AND DISCUSSION

88

3.1

Cyclic voltammetric studies of aflatoxins

88

3.1.1

89

Cathodic and anodic cyclic voltammetric of aflatoxins

xi 3.2

Differential pulse cathodic stripping voltammetry

102

of AFB2 3.2.1 Optimisation of conditions for the stripping

104

analysis 3.2.1.1 Effect of pH and type of supporting

104

electrolyte 3.2.1.2 Optmisation of instrumental conditions

117

3.2.1.2a Effect of scan rate

118

3.2.1.2b Effect of accumulation time

119

3.2.1.2c Effect of accumulation

120

potential

3.2.2

3.2.1.2d Effect of initial potential

121

3.2.1.2e Effect of pulse amplitude

122

Analysis of aflatoxins

127

3.2.2.1 Calibration curves of aflatoxins and

129

validation of the proposed method

3.3

3.2.2.1a Calibration curve of AFB2

129

3.2.2.1b Calibration curve of AFB1

134

3.2.2.1c Calibration curve of AFG1

137

3.2.2.1d Calibration curve of AFG2

140

3.2.2.2 Determination of limit of detection

143

3.2.2.3 Determination of limit of quantification

147

3.2.2.4 Inteference studies

150

Square-wave stripping voltammetry (SWSV) of

157

aflatoxins 3.3.1

SWSV determination of AFB2

158

3.3.1.1 Optimisation of experimental and

159

instrumental SWSV parameters 3.3.3.1a Influence of pH of BRB

159

3.3.3.1b Effect of instrumental

160

variables

xii

3.4

3.3.2 SWSV determination of other aflatoxins

166

3.3.3

168

Calibration curves and method validation

Stability studies of aflatoxins

175

3.4.1

10 ppm aflatoxin stock solutions

175

3.4.2

1 ppm aflatoxins in BRB at pH 9.0

179

3.4.2.1 Month to month stability studies

179

3.4.2.2 Hour to hour stability studies

181

3.4.2.3 Stability studies in different pH

186

of BRB 3.4.2.4 Stability studies in 1.0 M HCl and

191

1.0 M NaOH 3.5

4

Voltammetric analysis of aflatoxins in real samples

192

3.5.1 Study on the extraction techniques

193

3.5.2

Analysis of blank

194

3.5.3

Recovery studies of aflatoxins in real samples

196

3.5.4

Analysis of aflatoxins in real samples

199

CONCLUSIONS AND RECOMMENDATIONS

204

4.1

Conclusions

204

4.2

Recommendations

206

REFERENCES Appendices A – AM

208 255 - 317

xiii LIST OF TABLES

TABLE NO.

TITLE

PAGE

1.0

Scientific name for aflatoxin compounds

7

1.1

Chemical and physical properties of aflatoxin compounds

10

1.2

Summary of analysis methods used for determination of aflatoxins in various samples

19

1.3

Working electrode and limit of detection for modern polarographic and voltammetric techniques.

32

1.4

The application range of various analytical techniques and their concentration limits when compared with the requirements in different fields of chemical analysis

33

1.5

List of different type of working electrodes and its potential windows

36

1.6

Electroreducible and electrooxidisable organic functional groups

50

1.7

The characteristics of different type of electrochemical reaction.

53

1.8

Application of Square Wave Voltammetry technique

62

2.0

List of aflatoxins and their batch numbers used in this experiment

72

2.1

Injected volume of aflatoxins into eluate of groundnut and the final concentrations obtained in voltammetric cell

86

3.0

The dependence of current peaks of aflatoxins to their concentrations obtained by cathodic cyclic measurements in BRB at 9.0.

97

xiv 3.1

Effect of buffer constituents on the peak height of 2.0 µM AFB2 at pH 9.0. Experimental conditions are the same as Figure 3.21

109

3.2

Compounds reduced at the mercury electrode

116

3.3

Optimum parameters for 0.06 µM and 2.0 µM AFB2 in BRB at pH 9.0.

126

3.4

The peak height and peak potential of aflatoxins obtained by optimised parameters in BRB at pH 9.0 using DPCSV technique.

127

3.5

Peak height (in nA) obtained for intra-day and inter-day precision studies of 0.10 µM and 0.20 µM by the proposed voltammetric procedure (n=8).

131

3.6

Mean values for recovery of AFB2 standard solution (n=3).

132

3.7

Influence of small variation in some of the assay condition of the proposed procedure on its suitability and sensitivity using 0.10 µM AFB2.

133

3.8

Results of ruggedness test for proposed method using 0.10 µM AFB2.

134

3.9

Peak height (in nA) obtained for intra-day and inter-day precision studies of 0.10 µM and 0.20 µM AFB1 by proposed voltammetric procedure (n=5).

136

3.10

Mean values for recovery of AFB1 standard solution (n=3).

137

3.11

Peak height (in nA) obtained for intra-day and inter-day precision studies of 0.10 µM and 0.20 µM AFG1 by proposed voltammetric procedure (n=5).

139

3.12

Mean values for recovery of AFG1 standard solution (n=3).

139

3.13

Peak height (in nA) obtained for intra-day and inter-day precision studies of 0.10 µM and 0.20 µM AFG2 by proposed voltammetric procedure.

141

xv 3.14

Mean values for recovery of AFG2 standard solution (n=5).

142

3.15

Peak height and peak potential of 0.10 µM aflatoxins obtained by BAS and Metrohm voltammetry analysers under optimised operational parameters for DPCSV method.

143

3.16

Analytical parameters for calibration curves for AFB1,AFB2, AFG1 and AFG2 obtained by DPCSV technique using BRB at pH 9.0 as the supporting electrolyte.

145

3.17

LOD values for determination of aflatoxins obtained by various methods.

148

3.18

LOQ values for determination of aflatoxins obtained by various methods.

149

3.19

Peak current and peak potential for all aflatoxins obtained by SWSV in BRB at pH 9.0 (n=5).

167

3.20

Analytical parameters for calibration curves for AFB1, AFB2, AFG1 and AFG2 obtained by SWSV technique in BRB pH 9.0 as the supporting electrolyte.

172

3.21

Result of reproducibility study (intra-day and interday measurements) for 0.1 µM aflatoxins in BRB at pH 9.0 obtained by SWSV method.

173

3.22

Application of the proposed method in evaluation of the SWSV method by spiking the aflatoxin standard solutions.

174

3.23

Average concentration of all aflatoxins within a year stability studies.

175

3.24

The peak current and peak potential of 10 ppb AFB2 in presence and absence of a blank sample.

196

3.25

Total aflatoxin contents in real samples which were obtained by DPCSV and HPLC techniques (average of duplicate analysis)

203

xvi LIST OF FIGURES

FIGURE NO.

TITLE

PAGE

1.0

Aspergillus flavus seen under an electron microscope.

4

1.1

Chemical structure of coumarin

6

1.2

Chemical structures of (a) AFB1, (b) AFB2, (c) AFG1, (d) AFG2, (e) AFM1 and (f) AFM2

8

1.3

Hydration of (a) AFB1 and (b) AFG1 by TFA produces (c) AFB2a and (d) AFG2a

9

1.4

Transformation of toxic (a) AFB1 to non-toxic (b) aflatoxicol A.

11

1.5

Major DNA adducts of AFB1; (a) 8,9-Dihydro-8(N7-guanyl)-9-hydroxy-aflatoxin B1 (AFB1-Gua) and (b) 8,9-Dihydro-8-(N5-Formyl-2’.5’.6’triamino-4’-oxo-N5-pyrimidyl)-9-hydroxy-Aflatoxin B1 (AFB1-triamino-Py)

13

1.6

Metabolic pathways of AFB1 by cytochrom P-450 enzymes; B1-epoxide = AFB1 epoxide, M1= aflatoxin M1, P1= aflatoxin P1 and Q1 = aflatoxin Q1.

14

1.7

A typical arrangement for a voltammetric electrochemical cell (RE: reference electrode, WE: working electrode. AE: auxiliary electrode)

34

1.8

A diagram of the Hanging Mercury Drop Electrode (HMDE)

37

1.9

A diagram of the Controlled Growth Mercury Electrode (CGME)

38

1.10

Cyclic voltammograms of (a) reversible, (b) irriversible and (c) quasireversible reaction at mercury electrode (O = oxidised form and R = reduced form)

52

xvii

1.11

The potential-time sequence in stripping analysis

55

1.12

Schematic drawing showing the Faradaic current and charging current versus pulse time course

59

1.13

Schematic drawing of steps in DPV by superimposing a periodic pulse on a linear scan

60

1.14

Waveform for square-wave voltammetry

61

2.0

BAS CGME stand (a) which is connected to CV-50W voltammetric analyser and interface with computer (b) for data processing

71

2.1

VA757 Computrace Metrohm voltammetric analyser with 663 VA stand (consists of Multi Mode (MME))

71

3.0

Cathodic peak current of 0.6 µM AFB1 in various pH of BRB obtained in cathodic cyclic voltammetry. Ei = 0, Elow = -1.5 V, Ehigh = 0 and scan rate = 200 mV/s)

89

3.1

Shifting of peak potential of AFB1 with increasing pH of BRB. Parameter conditions are the same as in Figure 3.0.

90

3.2

Mechanism of reduction of AFB1 in BRB at pH 6.0 to 8.0.

90

3.3

Mechanism of reduction of AFB1 in BRB at pH 9.0 to 11.0.

91

3.4

Cathodic cyclic voltammogram for 1.3 µM AFB1 obtained at scan rate of 200 mV/s, Ei = 0, Elow = -1.5 V and Ehigh = 0 in BRB solution at pH 9.0.

92

3.5

Cathodic cyclic voltammogram for 1.3 µM AFB2 in BRB solution at pH 9.0. All parameter conditions are the same as in Figure 3.4.

92

3.6

Cathodic cyclic voltammogram for 1.3 µM AFG1 in BRB solution at pH 9.0. All parameter conditions are the same as in Figure 3.4.

93

xviii 3.7

Cathodic cyclic voltammogram for 1.3 µM AFG2 in BRB solution at pH 9.0. All parameter conditions are the same as in Figure 3.4.

93

3.8

Effect of Ei to the Ip of 0.6 µM AFB1 in BRB at pH 9.0 obtained by cathodic cyclic voltammetry. All parameter conditions are the same as in Figure 3.4.

94

3.9

Effect of Ei to the Ip of 0.1 µM Zn2+ in BRB at pH 9.0 obtained by cathodic cyclic voltammetry. All parameter conditions are the same as in Figure 3.4.

94

3.10

Anodic cyclic voltammogram of 1.3 µM AFB2 obtained at scan rate of 200 mV/s, Ei = -1.5 V, Elow = -1.5 V and Ehigh = 0 in BRB at pH 9.0.

95

3.11

Effect of increasing AFB2 concentration on the peak height of cathodic cyclic voltammetrc curve in BRB at pH = 9.0. (1.30 µM, 2.0 µM, 2.70 µM and 3.40 µM). All parameter conditions are the same as in Figure 3.4.

96

3.12

Peak height of reduction peak of AFB2 with increasing concentration of AFB2. All parameter conditions are the same as in Figure 3.4.

96

3.13

Repetitive cathodic cyclic voltammograms of 1.3 µM AFB2 in BRB solution at pH 9.0. All experimental conditions are the same as in Figure 3.4.

98

3.14

Increasing Ip of 1.3 µM AFB2 cathodic peak obtained from repetitive cyclic voltammetry. All experimental conditions are the same as in Figure 3.4.

98

3.15

Peak potential of 1.3 µM AFB2 with increasing number of cycle obtained by repetitive cyclic voltammetry.

99

xix 3.16

Plot of log Ip versus log υ for 1.3 µM AFB2 in BRB solution at pH 9.0. All experimental conditions are the same as in Figure 3.4.

100

3.17

Plot of Ep versus log υ for 1.3 µM AFB2 in BRB solution at pH 9.0.

101

3.18

Plot of Ip versus υ for 1.3 µM AFB2 in BRB solution at pH 9.0. All parameter conditions are the same as in Figure 3.4.

102

3.19

DPCS voltammograms of 1.0 µM AFB2 (Peak I) in BRB at pH 9.0 (a) at tacc = 0 and 30 s. Other parameter conditions; Ei = 0, Ef = -1.50 V, Eacc = 0, υ =50 mV/s and pulse amplitude = 100 mV. Peak II is the Zn peak.

103

3.20

DPCS voltammograms of 2.0 µM AFB2 in BRB in BRB (Peak I) at different pH values; 6.0, 7.0, 8.0, 9.0, 11.0and 13.0. Other parameter conditions; Ei = 0, Ef =-1.50 V, Eacc = 0, υ = 50 mV/s and pulse amplitude =100 mV. Peak II is the Zn peak.

104

3.21

Dependence of the Ip for AFB2 on the pH of 0.04 M BRB solution. AFB2 concentration: 2.0 µM, Ei =0, Ef = -1.5 V, E acc = 0, tacc = 30 sec, υ = 50 mV/s and pulse amplitude = 100 mV.

105

3.22

Ip of 2.0 µM AFB2 obtained in BRB (a) at pH from 9.0 decreases to 4.0 and re-increase to 9.0 and (b) at pH from 9.0 increase to 13.0 and re-decrease to 9.0.

106

3.23

UV-VIS spectrums of 1 ppm AFB2 in BRB at pH (a) 6.0, (b) 9.0 and (c) 13.0.

107

3.24

Opening of lactone ring by strong alkali caused no peak to be observed for AFB2 in BRB at pH 13.0.

108

3.25

Ip of 2.0 µM AFB2 in different concentration of BRB at pH 9.0. Experimental conditions are the same as in Figure 3.20.

109

3.26

Ip of 2.0 µM AFB2 in different pH and concentrations of BRB.

110

xx

3.27

DPCS voltammograms of 2.0 AFB2 (Peak I) in (a) 0.04 M, (b) 0.08 M and (c) 0.08 M BRB at pH 9.0 as the blank. Ei = 0 V, Ef = -1.5 V, Eacc = 0 V, tacc = 30 sec, υ = 50mV/s and pulse amplitude = 100 mV. Peak II is the Zn peak.

110

3.28

Chemical structures of (a) 2,3-dihydrofuran, (b) tetrahydrofuran and (c) coumarin.

111

3.29

Voltammograms of 2,3-dihydrifuran (peak I) for concentrations from (b) 0.02 to 0.2 µM in (a) BRB at pH 9.0.

112

3.30

Dependence of Ip of coumarin to its concentrations

112

3.31

Voltammograms of coumarin at concentration of (b) 34 µM, (c) 68 µM, (d) 102 µM, (e) 136 µM and (f) 170 µM in (a) BRB at pH 9.0.

113

3.32

Chemical structures of (a) cortisone and (b) testosterone.

114

3.33

Chemical structures of (a) digoxin and (b) digitoxin

115

3.34

Effect of pH of BRB solution on the Ep for AFB2. AFB2 concentration: 2.0 µM. Ei = 0, Ef = -1.5 V, Eacc = 0, t acc = 30 s and υ = 50 mV/s.

117

3.35

Effect of various υ to the (a) Ip and (b) Ep of 2.0 µM AFB2 peak in BRB at pH 9.0. Ei = 0, Ef = 1.50 V, Eacc = 0, t acc = 15 s and pulse amplitude = 100 mV.

118

3.36

Effect of tacc on (a) Ip and (b) Ep of 2.0 µM AFB2 peak in BRB at pH 9.0. Ei = 0, Ef = 1.50 V, Eacc = 0, υ = 40 mV/s and pulse amplitude = 100 mV.

119

3.37

The relationship between (a) Ip and (b) Ep with Eacc for 2.0 µM AFB2 in BRB at pH 9.0. Ei = 0, Ef = -1.5 V, tacc = 15 s, υ = 40 mV/s and pulse amplitude = 100 mV.

120

3.38

Effect of Ei on (a) Ip and (b) Ep of 2.0 µM AFB2 in BRB at pH 9.0. Ef = 1.50 V, Eacc = -0.80 V, tacc = 40 s,υ = 40 mV/s and pulse amplitude = 100 mV.

121

xxi 3.39

Effect of pulse amplitude on (a) Ip and (b) Ep of 2.0 µM AFB2 in BRB at pH 9.0. Ei = -1.0 V, Ef = 1.50 V, Eacc = -0.80 V, tacc = 40 s and υ = 40 mV/s.

122

3.40

Effect of Eacc on Ip of 0.06 µM AFB2. Ei = -1.0 V, Ef = -1.50 V, tacc = 40 s, υ = 40 mV/s and pulse amplitude = 100 mV.

123

3.41

Effect of tacc on (a) Ip and (b) Ep of 0.06 µM AFB2 in BRB at pH 9.0. Ei = -1.0 V, Ef = -1.50 V, Eacc = -0.6 V, υ = 40 mV/s and pulse amplitude = 100 mV.

124

3.42

The effect υ on (a) Ip and (b) Ep of 0.06 µM AFB2 In BRB at pH 9.0. Ei = -1.0 V, E f = -1.50 V, Eacc = -0.6 V, tacc = 80 s and pulse amplitude = 100 mV.

125

3.43

Voltammograms of 0.06 µM AFB2 obtained under (a) optimised and (b) unoptimised parameters in BRB at pH 9.0.

126

3.44

Voltammograms of 0.1 µM (a) AFB1, (b) AFG1, (c) AFB2 and (d) AFG2 in BRB at pH 9.0. Ei = 1.0 (except for AFG1 = -0.95 V), Ef = -1.4 V, Eacc = -0.6 V, tacc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV.

128

3.45

Voltammograms of (b) mixed aflatoxins in (a) BRB at pH 9.0 as the blank. Parameters condition: Ei = -0.95 V, Ef = -1.4 V, Eacc = -0.6 V, t acc = 80 s, υ = 50 mV/s and Pulse amplitude = 80 mV.

129

3.46

Increasing concentration of AFB2 in BRB at pH 9.0. The parameter conditions: Ei = -1.0 V, Ef = -1.4 V, Eacc =-0.6 V, t acc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV.

130

3.47

Linear plot of Ip versus concentration of AFB2 in BRB at pH 9.0. The parameter conditions are the same as in Figure 3.46.

130

3.48

Standard addition of AFB1 in BRB at pH 9.0. The parameter conditions: Ei = -1.0 V, Ef = -1.4 V, Eacc =-0.8 V, t acc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV.

135

xxii 3.49

Linear plot of Ip versus concentration of AFB1 in BRB at pH 9.0. The parameter conditions are the same as in Figure 3.48.

135

3.50

Effect of concentration to Ip of AFG1 in BRB at pH 9.0. Ei = -0.95 V, Ef = -1.40 V, Eacc =-0.8 V, tacc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV.

138

3.51

Linear plot of Ip versus concentration of AFG1 in BRB at pH 9.0. The parameter conditions are the same as in Figure 3.50.

138

3.52

Effect of concentration to Ip of AFG2 in BRB at pH 9.0. Ei = -1.0 V, Ef = -1.40 V, Eacc =-0.8 V, tacc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV.

140

3.53

Linear plot of Ip versus concentration of AFG2 in BRB at pH 9.0. The parameter conditions are the same as in Figure 3.52.

141

3.54

Voltammograms of 0.1 µM (a) AFB1, (b) AFB2, (c) AFG1 and (d) AFG2 in BRB at pH 9.0 (d) obtained by 747 VA Metrohm. Ei = -1.0 V (except for AFG1 = -0.95 V), Ef = -1.4 V, Eacc = -0.6 V, tacc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV.

144

3.55

Ip of all aflatoxins with increasing concentration of Zn 2+ up to 1.0 µM.

150

3.56

Voltammograms of (i) 0.1µM AFB2 and (ii) AFB2-Zn 151 complex with increasing concentration of Zn2+ (a = 0, b = 0.75 µM, c = 1.50 µM, d = 2.25 µM and e = 3.0 µM). Blank = BRB at pH 9.0. Experimental conditions; Ei = -1.0 V, Ef = -1.40 V, Eacc = -0.6 V, tacc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV.

3.57

Ip of all aflatoxins after reacting with increasing concentration of Zn2+ in BRB at pH 3.0. Measurements were made in BRB at pH 9.0 within 15 minutes of reaction time. Ei = -1.0 V (except for AFG1 = -0.95 V), Ef = -1.40 V, Eacc = -0.6 V, tacc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV.

151

xxiii 3.58

Absorbance of all aflatoxins with increasing concentration of Zn2+ in BRB at pH 3.0 within 15 minutes of reaction time

152

3.59

Voltammograms of 0.1µM AFB2 with increasing concentration of Zn2+ (from 0.10 to 0.50 µM) in BRB at pH 9.0. Ei = -0.25 V, Ef = -1.4 V, Eacc = -0.6 V, tacc = 80 s, υ = 50 mV/s and pulse amplirude = 80 mV. .

153

3.60

Chemical structure of ascorbic acid.

153

3.61

Ip of all aflatoxins with increasing concentration of ascorbic acid up to 1.0 µM. Concentrations of all aflatoxins are 0.1µM.

154

3.62

Voltammograms of 0.1µM AFB2 with increasing concentration of ascorbic acid.

154

3.63

Ip of all aflatoxins with increasing concentration of β-cyclodextrin up to 1.0 µM.

155

3.64

Voltammograms of 0.1µM AFB2 with increasing concentration of β-cyclodextrin.

156

3.65

Chemical structure of L-cysteine.

156

3.66

Ip of all aflatoxins with increasing concentration of cysteine up to 1.0 µM.

157

3.67

Voltammograms of 0.1 µM AFB2 obtained by (a) DPCSV and (b) SWSV techniques in BRB at pH 9.0. Parameters for DPCSV: Ei = -1.0 V, Ef = -1.40V, Eacc = -0.6 V, t acc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV and for SWSV: Ei = -1.0 V, Ef = -1.40V, Eacc = -0.6 V, tacc = 80 s, frequency = 50 Hz, voltage step = 0.02 V, amplitude = 80 mV and υ = 1000 mV/s.

158

3.68

Influence of pH of BRB on the Ip of 0.10 µM AFB2 using SWSV technique. The instrumental Parameters are the same as in Figure 3.67.

159

3.69

Voltammograms of 0.1 µM AFB2 in different pH of BRB. Parameter conditions: Ei = -1.0 V, E f = -1.40 V, Eacc = -0.6 V, tacc = 80 s, voltage step =

160

xxiv 0.02 V, amplitude = 50 mV, frequency = 50 Hz and υ = 1000 mV/s. 3.70

Effect of Ei to the Ip of 0.10 µM AFB2 in BRB at pH 9.0.

160

3.71

Effect of Eaccto the Ip of 0.10 µM AFB2 in BRB at pH 9.0.

161

3.72

Relationship between Ip of 0.10 µM AFB2 while increasing tacc.

162

3.73

Effect of frequency to the Ip of 0.10 µM AFB2 in BRB at pH 9.0.

162

3.74

Linear relatioship between Ip of AFB2 and square root of frequency.

163

3.75

Influence of square-wave voltage step to Ip of 0.10 µM AFB2.

163

3.76

Influence of square-wave amplitude to Ip of 0.10 µM AFB2.

164

3.77

Relationship of SWSV Ep of AFB2 with increasing amplitude.

164

3.78

Ip of 0.10 µM AFB2 obtained under (a) non-optimised and (b) optimised SWSV parameters compared with that obtained under (c) optimised DPCSV parameters.

165

3.79

Voltammograms of 0.10 µM AFB2 obtained under (a) non-optimised and (b) optimised SWSV parameters compared with that obtained under (c) optimised DPCSV parameters.

166

3.80

Ip of 0.10 µM aflatoxins obtained using two different stripping voltammetric techniques under their optimum paramter conditions in BRB at pH 9.0.

167

3.81

Voltammograms of (i) AFB1, (ii) AFB2, (iii) AFG1 and (iV) AFG2 obtained by (b) DPCSV compared with that obtained by (c) SWSV in (a) BRB at pH 9.0.

168

3.82

SWSV voltammograms for different concentrations of AFB1 in BRB at pH 9.0. The broken line

169

xxv represents the blank: (a) 0.01 µM, (b) 0.025 µM, (c) 0.05 µM, (d) 0.075 µM, (e) 0.10 µM, (f) 0.125 µM and (g) 0.150 µM. Parameter conditions: Ei = -1.0 V, Ef = -1.4 V, Eacc = -0.8 V, t acc = 100 s, frequency = 125 Hz, voltage step = 0.03 V, pulse amplitude = 75 mV and scan rate = 3750 mV/s. 3.83

Calibration curve for AFB1 obtained by SWSV method.

170

3.84

Calibration curve for AFB2 obtained by SWSV method.

170

3.85

Calibration curve for AFG1 obtained by SWSV method.

170

3.86

Calibration curve for AFG2 obtained by SWSV method.

171

3.87

LOD for determination of aflatoxins obtained by two different stripping methods.

171

3.88

UV-VIS spectrums of 10 ppm of all aflatoxins in benzene: acetonitrile (98%) at preparation date.

176

3.89

UV-VIS spectrums of 10 ppm of all aflatoxins in benzene: acetonitrile (98%) after 6 months of storage time.

176

3.90

UV-VIS spectrums of 10 ppm of all aflatoxins in benzene: acetonitrile (98%) after 12 months of storage time.

177

3.91

UV-VIS spectrums of 10 ppm AFB1 (a) kept in the cool and dark conditions and (b) exposed to ambient conditions for 3 days.

178

3.92

UV-VIS spectrums of 1 ppm AFB1 in BRB solution prepared from (a) good and (b) damaged 10 ppm AFB1 stock solution.

178

3.93

UV-VIS spectrums of 1 ppm AFB1 in BRB solution prepared from (a) good and (b) damaged 10 ppm AFB1 stock solution after 2 week stored in the cool and dark conditions.

179

xxvi 3.94

Percentage of Ip of 0.10 µM aflatoxins in BRB at pH 9.0 at different storage time in the cool and dark conditions.

180

3.95

Percentage of Ip of all aflatoxins in BRB at pH 9.0 exposed to ambient conditions up to 8 hours of exposure time.

181

3.96

Reaction of 8,9 double bond furan rings in AFB1 with TFA, iodine and bromine under special conditions (Kok, et al., 1986).

182

3.97

Voltammograms of 0.10 µM AFB1 obtained in (a) BRB at pH 9.0 which were prepared from (b) damaged and (c) fresh stock solutions.

183

3.98

Ip of 0.10 µM AFB1 obtained in BRB at pH 9.0 from 0 to 8 hours in (a) light exposed and (b) light protected.

184

3.99

Ip of 0.10 µM AFB2 obtained in BRB at pH 9.0 from 0 to 8 hours in (a) light exposed and (b) light protected.

184

3.100

Ip of 0.10 µM AFG1 obtained in BRB at pH 9.0 from 0 to 8 hours in (a) light exposed and (b) light protected.

185

3.101

Ip of 0.10 µM AFG2 obtained in BRB at pH 9.0 from 0 to 8 hours in (a) light exposed and (b) light protected.

185

3.102

Peak heights of 0.10 µM aflatoxins in BRB at pH (a) 6.0, (b) 7.0, (c) 9.0 and (d) 11.0 exposed to ambient conditions up to 3 hours of exposure time.

187

3.103

Resonance forms of the phenolate ion (Heathcote, 1984).

187

3.104

Voltammograms of 0.10 µM AFB2 in BRB at pH 6.0 from 0 to 3 hrs exposure time.

188

3.105

Voltammograms of 0.10 µM AFB2 in BRB at pH 11.0 from 0 to 3 hrs of exposure time.

188

xxvii 3.106

Voltammograms of 0.10 µM AFG2 in BRB at pH 6.0 from 0 to 3 hrs of exposure time.

189

3.107

Voltammograms of 0.10 µM AFG2 in BRB at pH 11.0 from 0 to 3 hrs of exposure time.

189

3.108

Absorbance of 1.0 ppm aflatoxins in BRB at pH (a) 6.0, (b) 7.0, (c) 9.0 and (d) 11.0 from 0 to 3 hours of exposure time.

190

3.109

The peak heights of aflatoxins in 1.0 M HCl from 0 to 6 hours of reaction time.

191

3.110

The peak heights of aflatoxins in 1.0 M NaOH from 0 to 6 hours of reaction time.

192

3.111

Voltammograms of real samples after extraction by Technique (a) 1, (b) 2 and (c) 3 with addition of AFB1 standard solution in BRB at pH 9.0 as a blank.

193

3.112

Voltammograms of blank in BRB at pH 9.0 obtained by (a) DPCSV and (b) SWSV methods.

194

3.113

DPCSV (a) and SWSV (b) voltammograms of 10 ppb AFB2 (i) in present of blank sample (ii) obtained in BRB at pH 9.0 (iii) as the supporting electrolyte.

195

3.114

DPCSV voltammograms of real samples (b) added with 3 ppb (i), 9 ppb (ii) and 15 ppb (iii) AFB1 obtained in BRB at pH 9.0 (a) as the blank on the first day measurement.

197

3.115

Percentage of recoveries of (a) 3 ppb, (b) 9 ppb, (c) 15 ppb of all aflatoxins in real samples obtained by DPCSV methods for one to three days of measurements.

198

3.116

DPCSV voltammograms of real sample, S11 (b) with the addition of 10 ppb AFB1 (c) in BRB at pH 9.0 (a) as the blank. Parameter conditions: Eacc = -0.6 V, tacc = 80 s, scan rate = 50 mV/s and pulse amplitude = 80 mV.

199

xxviii 3.117

SWSV voltammograms of real sample, S11 (b) with the addition of 10 ppb AFB1 (c) in BRB at pH 9.0 (a) as the blank. Parameter conditions: Eacc = -0.8 V, tacc = 100 s, scan rate = 3750 mV/s, frequency = 125 Hz, voltage step = 0.03 V and pulse amplitude = 75 mV.

200

3.118

DPCSV voltammograms of real sample, S07 (b) with the addition of 10 ppb AFB1 (c) in BRB at pH 9.0 (a) as the blank. Parameter conditions: Eacc = -0.6 V, tacc = 80 s, scan rate = 50 mV/s and pulse amplitude = 80 mV.

200

3.119

SWSV voltammograms of real sample, S07 (b) with the addition of 10 ppb AFB1 (c) in BRB at pH 9.0 (a) as the blank. Parameter conditions: Eacc = -0.8 V, tacc = 100 s, scan rate = 3750 mV/s, frequency = 125 Hz, voltage step = 0.03 V and pulse amplitude = 75 mV.

201

3.120

DPCSV voltammograms of real sample, S10 (b) with the addition of 10 ppb AFB1 (c) in BRB at pH 9.0 (a) as the blank. Parameter conditions: Eacc = -0.6 V, tacc = 80 s, scan rate = 50 mV/s and pulse amplitude = 80 mV.

201

3.121

SWSV voltammograms of real sample, S10 (b) with the addition of 10 ppb AFB1 (c) in BRB at pH 9.0 (a) as the blank. Parameter conditions: Eacc = -0.8 V, tacc = 100 s, scan rate = 3750 mV/s, frequency = 125 Hz, voltage step = 0.03 V and pulse amplitude = 80 mV.

202

xxix ABBREVIATIONS

AAS

Atomic absorption spectrometry

Abs

Absorbance

ACP

Alternate current polarography

ACV

Alternate current voltammetry

AD

Amperometric detector

AdCSV

Adsorptive cathodic stripping voltammetry

AE

Auxiliary electrode

AFB1

Aflatoxin B1

AFB2

Aflatoxin B2

AFG1

Aflatoxin G1

AFG2

Aflatoxin G2

AFM1

Aflatoxin M1

AFM2

Aflatoxin M2

AFP1

Aflatoxin P1

AFQ1

Aflatoxin Q1

Ag/AgCl

Silver/silver chloride

ASV

Anodic stripping voltammetry

β-CD

β-cyclodextrin

BFE

Bismuth film electrode

BLMs

Bilayer lipid membranes

BRB

Britton Robinson Buffer

CA

Concentration of analyte

CE

Capillary electrophoresis

CGME

Controlled growth mercury electrode

CME

Chemically modified electrode

CPE

Carbon paste electrode

CSV

Cathodic stripping voltammetry

CV

Cyclic voltammetry

xxx DC

Direct current

DCP

Direct current polarography

DME

Dropping mercury electrode

DMSO

Dimethyl sulphonic acid

DNA

Deoxyribonucliec acid

DPCSV

Differential pulse cathodic stripping voltammetry

DPP

Differential pulse polarography

DPV

Differential pulse voltammetry

Eacc

Accumulation potential

Ei

Initial potential

Ef

Final potential

Ehigh

High potential

Elow

Low potential

Ep

Peak potential

ECS

Electrochemical sensing

Et4NH4 OH

Tetraethyl ammonium hydroxide

ELISA

Enzyme linked immunosorbant assay

FD

Fluorescence detector

FDA

Food and Drug Administration

GCE

Glassy carbon electrode

GC-FID

Gas chromatography with flame ionisation detector

HMDE

Hanging mercury drop electrode

HPLC

High performance liquid chromatography

HPTLC

High pressure thin liquid chromatography

IAC

Immunoaffinity chromatography

IACLC

Immunoaffinity column liquid chromatography

IAFB

Immunoaffinity fluorometer biosensor

IARC

International Agency for Research Cancer

Ic

Charging current

Id

Diffusion current

If

Faradaic current

xxxi Ip

Peak height

ICP-MS

Induced coupled plasma-mass spectrometer

IR

Infra red

IUPAC

International Union of Pure and Applied Chemistry

KGy

Kilogray

LD50

Lethal dose 50

LOD

Limit of detection

LOQ

Limit of quantification

LSV

Linear sweep voltammetry

MFE

Mercury film electrode

MS

Mass spectrometer

MECC

Micellar electrokinetic capillary chromatography

MOPS

3-(N-morpholino)propanesulphonic

MOSTI

Ministry of Science, Technology and Innovation

NP

Normal polarography

NPP

Normal pulse polarography

NPV

Normal pulse voltammetry

OPLC

Over pressured liquid chromatography

PAH

Polycyclic aromatic hydrocarbon

PLL

Poly-L-lysine

ppb

part per billion

ppm

part per million

PSA

Potentiometric stripping analysis

RDX

Hexahydro-1,3,5-trinitro-1,3,5-triazine

RE

Reference electrode

RIA

Radioimmunoassay

RNA

Ribonucleic acid

RSD

Relative standard deviation

S/N

Signal to noise ratio

SCE

Standard calomel electrode

SCV

Stair case voltammetry

xxxii SDS

Sodium dodecyl sulphate

SHE

Standard hydrogen electrode

SIIA

Sequential injection immunoassay

SMDE

Static mercury drop electrode

SPE

Solid phase extraction

SPCE

Screen printed carbon electrode

SWP

Square-wave polarography

SWV

Square-wave voltammetry

SWSV

Square-wave stripping voltammetry

SV

Stripping voltammetry

tacc

Accumulation time

TBS

Tris buffered saline

TEA

Triethylammonium

TLC

Thin layer chromatography

TFA

Trifluoroacetic acid

UME

Ultra microelectrode

ν

Scan rate

v/v

Volume per volume

UVD

Ultraviolet-Visible detector

UV-VIS

Ultraviolet-Visible

WE

Working electrode

WHO

World Health Organisation

λmax

Maximum wavelenght

εmax

Maximum molar absorptivity

xxxiii LIST OF APPENDICES

APPENDIX

TITLE

PAGE

A

Relative fluorescence of aflatoxins in different solvent s

255

B

UV spectra of the principal aflatoxins (in methanol)

256

C

Relative intensities of principal bands in the IR spectra of the aflatoxins

257

D

Calculation of concentration of aflatoxin stock solution

258

E

Extraction procedure for aflatoxins in real samples

259

F

Calculation of individual aflatoxin in groundnut samples.

260

G

Cyclic voltammograms of AFB1, AFB2 and AFG2 with increasing of their concentrations.

262

H

Dependence if the peak heights of AFB1, AFG1 and AFG2 on their concentrations.

264

I

Repetitive cyclic voltammograms and their peak heights of AFB1, AFG1 and AFG2 in BRB at pH 9.0

266

J

Plot Ep – log scan rate for the reduction of AFB1, AFG1 and AFG2 in BRB at pH 9.0

270

K

Plot of peak height versus scan rate for 1.3 µM of AFB1, AFG1 and AFG2 in BRB at pH 9.0

272

L

Voltammograms of AFB2 with increasing concentration.

274

M

Voltammograms of 0.1 µM and 0.2 µM AFB2 obtained on the same day measurements

275

N

Voltammograms of AFB2 at inter-day measurements

276

xxxiv

O

F test for robustness and ruggedness tests

278

P

Voltammograms of AFB1 with increasing concentration

280

Q

Voltammograms of AFG1 with increasing concentration

281

R

Voltammograms of AFG2 with increasing concentration

282

S

LOD determination according to Barek et al. (2001a)

283

T

LOD determination according to Barek et al. (1999)

286

U

LOD determination according to Zhang et al. (1996)

287

V

LOD determination according to Miller and Miller (1993)

288

W

ANOVA test

290

X

Peak height of aflatoxins in presence and absence of PLL

292

Y

SWSV voltammograms of AFB1, AFB2, AFG1 and AFG2 in BRB at pH 9.0

293

Z

SWSV voltammograms of 0.10 µM AFB1, AFB2, AFG1 and AFG2 in BRB at pH 9.0

295

AA

UV-VIS spectrums of 10 ppm AFB1, AFB2, AFG1 and AFG2 stock solutions

297

AB

Voltammograms of AFB1, AFB2, AFG1 and AFG2 obtained from 0 to 6 months of storage time in the cool and dark conditions.

299

AC

Voltammograms of AFB1, AFB2, AFG1 and AFG2 in BRB at pH 9.0 from 0 to 8 hours of exposure time.

302

AD

UV-VIS spectrums of AFB2 in BRB at pH 6.0 and 11.0.

305

xxxv AE

Voltammograms of AFB1 and AFG1 in 1.0 M HCl and 1.0 M NaOH

307

AF

DPCSV voltammograms of real samples added with various concentrations of AFG1

309

AG

SWSV voltammograms of real samples added with various concentrations of AFB1.

310

AH

Percentage of recoveries of various concentrations of all aflatoxins (3.0 and 9.0 ppb) in real samples obtained by SWSV method.

311

AI

Calculation of percentage of recovery for 3.0 ppb AFG1 added into real samples.

312

AJ

HPLC chromatograms of real samples: S10 and S07

313

AK

Calculation of aflatoxin in real sample, S13

314

AL

List of papers presented or published to date resulting from this study.

315

AM

ICP-MS results for analysis of BRB at pH 9.0

317

CHAPTER I

LITERATURE REVIEW

1.1

Overview Humans are continuously exposed to varying amounts of chemicals that have

been shown to have carcinogenic or mutagenic properties in environmental systems. Exposure can occur exogenously when these agents are present in food, air or water, and also endogenously when they are products of metabolism or pathophysiologic states such as inflammation. Great attention is focused on environmental health in the past two decades as a consequence of the increasing awareness over the quality of life due to major environment pollutants that affect it. Studies have shown that exposure to environmental chemical carcinogens have contributed significantly to cause human cancers, when exposures are related to life style factors such as diet (Wogan et al., 2004). The contamination of food is part of the global problem of environmental pollution. Foodstuffs have been found contaminated with substances having carcinogenic, mutagenic, teratogenic and allergenic properties. As these substances can be supplied with food throughout the entire life-time of a person, it is necessary to deal with the chronic action of trace amounts of such substances. Hence the systematic determination of the foreign substances in nutritional products and feedstock plays an important role. The determination of trace impurities presents considerable difficulties owing to the fact that food is a complex system containing thousands of major and minor compounds (Nilufer and Boyacio, 2002). Increasing environment pollution by toxic substances such as toxic metals, organometallic and organic pollutants in air,

2 water, soil and food, calls for reliable analytical procedures for their control in environmental samples which needs reliable and sensitive methods (Fifield and Haines, 2000). The choice of the method of analysis depends on the sample, the analyte to be assayed, accuracy, limit of detection, cost and time to complete the analysis (AboulEneim et a., 2000). For development of this method, emphasis should be on development of simplified, cost-effective and efficient method that complies with the legislative requirements (Stroka and Anklam, 2002; Enker, 2003). The widespread occurrence of aflatoxins producing fungi in our environment and the reported naturally occurring of toxin in a number of agricultural commodities has led the investigator to develop a new method for aflatoxin analysis (Creepy, 2002). An accurate and sensitive method of analysis is therefore required for the determination of these compounds in foodstuffs that have sustained mould growth. Numerous articles concerning methods for determination of aflatoxins have been published. However, with regard to electroanalytical technique, only one method of determination was reported using the differential pulse pulse polarographic (DPP) technique which was developed by Smyth et al. (1979). In this experiment, the obtained limit of detection of aflatoxin B1 was 25 ppb which was higher as compared to the common amount of aflatoxin in contaminated food samples which is 10 ppb as reported by Pare (1997) or even less. In Malaysia, the regulatory limit for total aflatoxins in groundnut is 15 ppb. The regulatory for other foods and milk is 10 ppb and 0.05 ppb respectively (Malaysian Food Act, 1983).

1.2

AFLATOXINS

1.2.1 Aflatoxins in General Aflatoxins are a group of heterocyclic, oxygen-containing mycotoxins that possess the bisdifuran ring system. It was discovered some 43 years ago in England

3 following a poisoning outbreak causing 100,000 turkeys death (Miller, 1987 and Cespedez and Diaz, 1997). The aflatoxins are the most widely distributed fungal toxins in food. The occurrence of the aflatoxins in food products demonstrated that the high levels of aflatoxins are significant concern both for food traders and food consumers (Tozzi et al. 2003; Herrman, 2004; Haberneh, 2004). Aflatoxin is a by-product of mold growth in a wide range of agriculture commodities such as peanuts (Urano et al., 1993), maize and maize based food (Papp et al., 2002; Mendez-Albores et al., 2004), cottonseeds (Pons and Franz, 1977), cocoa (Jefferey et al., 1982), coffee beans (Batista et al., 2003), medical herbs ( Reif and Metzger, 1998; Rizzo et al., 2004), spices (Erdogen, 2004; Garner et al., 1993; Akiyama et al., 2001; Aziz et al., 1998), melon seeds (Bankole et al., 2004) and also in human food such as rice (Shotwell et al., 1966; Begum and Samajpati, 2000), groundnut (Bankole et al., 2005), peanut products ( Patey et al., 1990), corn ( Shotwell and Goulden, 1977; Urano et al., 1993 ), vegetable oil (Miller et al., 1985), beer (Scott and Lawrence, 1997), dried fruits ( Abdul Kadar et al., 2004, Arrus et al. (2004), milk and dairy products (Kamkar, 2004; Aycicek et al. 2005; Sarimehmetoglu et al., 2004; Martin and Martin, 2004 ). Meat and meat products are also contaminated with alfatoxins when farm animals are fed with aflatoxin contaminated feed (Miller, 1987 and Chiavaro et al., 2001). The molds that are major producers of aflatoxin are Aspergillus flavus (Bankole et al. 2004) and Aspergillus parasiticus (Begum and Samajpati, 2000; Setamou et al., 1997; Erdogen, 2004; Gourama and Bullerman, 1995). Aspergillus flavus, which is ubiquitous, produces B aflatoxins (Samajphati, 1979) while Aspergillus parasiticus, which produces both B and G aflatoxins, has more limited distribution (Garcia-Villanova et al., 2004). A picture of Aspergillus flavus seen under an electron microscope is shown in Figure 1.0. Black olive is one of the substrate for Aspergillus parasiticus growth and aflatoxin B1 production as reported by Leontopoulos et al., (2003). Biosynthesis of aflatoxins by this fungi depends on the environmental condition such as temperature and humidity during crop growth and storage (Leszczynska et al., 2000; Tarin et al., 2004

4 and Pildain et al., 2004). The optimum temperatures for aflatoxins growth are 27.84 0 C and 27.30 0 C at pH=5.9 and 5.5 respectively.

Figure 1.0:

Aspergillus flavus seen under an electron microscope

Before harvest, the risk for the development of aflatoxins is greatest during major drought (Turner et al., 2005). When soil moisture is below normal and temperature is high, the number of Aspergillus spores in the air increases. These spores infect crops through areas of damage caused by insects and inclement weather. Once infected, plant stress occurs, which favor the production of aflatoxins. Fungal growth and aflatoxins contamination are the sequence of interactions among the fungus, the host and the environment. The appropriate combination of water stress, high temperature stress and insect damage of the host plant are major determining factors in mold infestation and toxin production (Faraj et al., 1991; Koehler, 1985; Park and Bullerman, 1983). Additional factors such as heat treatment, modified-atmosphere packaging or the presence of preservative, also contribute in increasing growth rate of the aflatoxins. Farmers have minimal control over some of these environmental factors. However appropriate pre-harvest and post-harvest management and good agricultural practice, including crop rotation, irrigation, timed planting and harvesting and the use of pesticides are the best methods for preventing or controlling aflatoxins contamination

5 (Turner et al., 2005). Timely harvesting could reduce crop moisture to a point where the formation of the mould would not occur. For example harvesting corn early when moisture is above 20 percent and then quickly drying it to a moisture level of at least 15 percent will keep the Aspergillus flavus from completing its life cycle, resulting in lower aflatoxin concentration. Aflatoxins are to be found in agricultural products as a consequence of unprosperous storage conditions where humidity of 70 -90 % and a minimum temperature of about 10° C. Commodities that have been dried to about 12 to 0.5 % moisture are generally considered stable, and immune to any risk of additional aflatoxins development. Moreover, the minimum damage of shells during mechanized harvesting of crop reduces significantly the mould contamination. Biocontrol of aflatoxin contamination is another way to reduce this contamination. The natural ability of many microorganisms including bacteria, actinomycetes, yeasts, moulds and algae has been a source for bacteriological breakdown of mycotoxins. The most active organism such as Flavobacterium aurantiacum which in aqueous solution can take up and metabolise aflatoxins B1, G1 and M1 (Smith and Moss, 1985). Production of aflatoxins is greatly inhibited by propionic acid as revealed by Molina and Gianuzzi (2002) when they studied the production of aflatoxins in solid medium at different temperature, pH and concentration of propionic acid. It also can be inhibited by essential oil extracted from thyme as found by Rasooli and Abnayeh (2004). Other chemicals that can inhibit the growth of this fungus are ammonia, copper sulphate and acid benzoic (Gowda et al., 2004). 1.2.2

Chemistry of Aflatoxins Aflatoxins can be classified into two broad groups according to chemical

structure which are difurocoumarocyclopentenone series and ifurocoumarolactone (Heathcote, 1984). They are highly substituted coumarin derivatives that contain a fused dihydrofuran moiety. The chemical structure of coumarin is shown in Figure 1.1.

6 O

O

Figure 1.1

Chemical structure of coumarin

There are six major compounds of aflatoxin such as aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG2), aflatoxin G2 (AFG2), aflatoxin M1 (AFM1) and aflatoxin M2 (AFM2) (Goldblatt, 1969). The former four are naturally found aflatoxins and the AFM1 and AFM2 are produced by biological metabolism of AFB1 and AFB2 from contaminated feed used by animals. They are odorless, tasteless and colorless. The scientific name for these aflatoxin compounds are listed in Table 1.0. Aflatoxins have closely similar structures and form a unique group of highly oxygenated, naturally occurring heterocyclic compounds. The chemical structures of these aflatoxins are shown in Figure 1.2. The G series of aflatoxin differs chemically from B series by the presence of a β-lactone ring, instead of a cyclopentanone ring. Also a double bond that may undergo reduction reaction is found in the form of vinyl ether at the terminal furan ring in AFB1 and AFG1 but not in AFB2 and AFG2. However this small difference in structure at the C-2 and C-3 double bond is associated with a very significant change in activity, whereby AFB1 and AFG1 are carcinogenic and considerably more toxic than AFB2 and AFG2. The dihydrofuran moiety in the structure is said to be of primary importance in producing biological effects. Hydroxylation of the bridge carbon of the furan rings for AFM1 does not significantly alter the effects of the compounds. The absolute configuration of AFB2 and AFG2 follows from the fact that it is derived from the reduction of AFB1 and AFG1 respectively. AFB is the aflatoxin which produces a blue color under ultraviolet while AFG produces the green color. AFM produces a blue-violet fluorescence while AFM2 produces a violet fluorescence (Goldblatt, 1969). Relative fluorescence of aflatoxins in several organic solvents are shown in Appendix A (White and Afgauer, 1970). The

7 Table 1.0

Scientific name for aflatoxin compounds

Aflatoxin B1

2,3,6a,9a-tetrahydro-4-methoxycyclopenta[c]

(AFB1)

furo[3’,2’:4,5]furo[2,3-h][l] benzopyran-1,11-dione

Aflatoxin B2

2,3,6a,8,9,9a-Hexahydro-4-methoxycyclopenta[c]

(AFB2)

furo[3’,2’:4,5]furo[2,3-h][l] benzopyran-1,11-dione

Aflatoxin G1 3,4, 7a,10a-tetrahydro-5-methoxy-1H, 12H (AFG1)

furo[3’,2’:4,5]furo[2,3-h]pyrano[3,4-c]l]- benzopyran1,12-dione

Aflatoxin G2 3,4,7a,8,9,10, 10a-Hexahydro-5-methoxy-1H,12H(AFG2)

furo[3’,2’:4,5]furo[2,3-h]pyrano[3,4-c][l]- benzopyran1,12-dione

Aflatoxin

4-Hydroxy AFB1

M1 (AFM1) Aflatoxin

4-Hydroxy AFB2

M2

natural fluorescence of aflatoxins arises from their oxygenated pentaheterocyclic structure. The fluorescence capacity of AFB2 and AFG2 is ten times larger than that of AFB1 and AFG1, probably owing to the structural difference, namely double bond on the furanic ring. Such a double bond seems to be very important for the photophysical

8

O

O

O

O

O

O

O

O

O

O

O

O

(a)

(b)

O

O

O

O

O

O

O

O

O

O

O

O

O

O

(c) O

(d) O O

O

O O

OH

OH

O

O

O

O

O

O

(e) Figure 1.2

(f)

Chemical structures of (a) AFB1, (b) AFB2, (c) AFG1, (d) AFG2,

(e) AFM1 and (f) AFM2. properties of these derivatives measured just after spectroscopic studies (Cepeda et al., 1996). The excitation of the natural fluorescence of AFB1 and AFG1 can be promoted

9

in many different ways such as post-column iodination (Tuinstra and Haasnoot, 1983; Davis and Diener, 1980), post-column bromination (Kok et al.,1986; Kok, 1994 ; Versantroort et al., 2005 ), use of cyclodextrin compound (Cepeda et al., 1996; Chiavaro et al., 2001; Franco et al., 1998) and trifluoroacetic acid, TFA (Stack and Pohland,

1975; Takahashi, 1977a; Haghighi et al., 1981; Nieduetzki et al., 1994 ). AFB1 and AFG1 form hemiacetals, AFB2a and AFG2a when reacted with acidic solution such as triflouroacetic acid (TFA) as represented in Figure 1.3 (Joshua, 1993). The hydroxyaflatoxins are unstable and tend to decompose to yellow products in the presence of air, light and alkali. Their UV and visible spectra are similar to those of the major aflatoxins.

O

O O

O

O O

TFA HO O

O

O

O

O

O

(a) O

(c)

O

O

O O

O

TFA

O

O

HO

O

O O

O

(b) Figure 1.3

O

O

(d)

Hydration of AFB1 (a) and AFG1 (b) by TFA produces AFB2a (c) and

AFG2a (d) (Joshua, 1993). The close relationship between AFB1, AFG1, AFB2a and AFG2a was shown by the similarities in their IR and UV spectra. The main difference between AFB2a and AFG2a with AFB1 and AFG1 are found in the IR spectra, where an additional band at 3620 cm-1

10 indicates the presence of a hydroxyl group in AFB2a and AFG2a. The absence of bands at 3100, 1067 and 722 cm-1 (which arise in AFB1 and AFG1 from the vinyl ether group) indicates that the compounds are hydroxyl derivatives of AFB2 and AFG2. Some chemical and physical properties of aflatoxin compounds are listed in Table 1.1 (Heathcote and Hibbert, 1978; Weast and Astle, 1987). The close relationship between these aflatoxins was shown by the similarities in their UV and IR spectra as shown in Appendix B and C respectively.

Table 1.1

Chemical and physical properties of aflatoxin compounds

AFB1

AFB2

AFG1

AFG2

Molecular formula

C17H12O6

C17H14O6

C17H12O7

C17H14O7

Molecular weight

312

314

328

330

Crystals

Pale yellow

Pale yellow

Colorless

Colorless

Melting point (°C)

268.9

286.9

244.6

237.40

Fluorescence under UV light

Blue

Blue

Green

Green

Solubility

Soluble in water and polar organic solvent. Normal solvents are: methanol, water: acetonitrile (9:1), trifluoroacetic acid, methanol: 0.1N HCl (4:1), DMSO and acetone

Other properties

Odorless, colorless and tasteless in solution form. Incompatible with strong acids, strong oxidising agents and strong bases. Soluble in water, DMSO, 95% acetone or ethanol for 24 hours under ambient temperature

STRIPPING VOLTAMMETRIC METHODS FOR THE DETERMINATION OF AFLATOXIN COMPOUNDS

MOHAMAD HADZRI BIN YAACOB

UNIVERSITI TEKNOLOGI MALAYSIA

BAHAGIAN A – Pengesahan Kerjasama * Adalah disahkan bahawa projek penyelidikan tesis ini telah dilaksanakan melalui kerjasama antara _____________________ dengan _________________________ Disahkan oleh: Tandatangan

: ..........................................................

Nama

: ..........................................................

Tarikh : ..........................

Jawatan:........................................................... (Cop rasmi) * Jika penyediaan tesis/projek melibatkan kerjasama. BAHAGIAN B – Untuk Kegunaan Pejabat Sekolah Pengajian Siswazah Tesis ini telah diperiksa dan diakui oleh: Nama dan Alamat Pemeriksa Luar

:

Prof. Dr. Noor Azhar Bin Mohd Shazili Pengarah Institut Oseanografi, Kolej Universiti Sains dan Teknologi Malaysia, Mengabang Telipot 21030 Kuala Terengganu

Nama dan Alamat Pemeriksa Dalam I

:

Prof. Madya Dr. Razali Bin Ismail Fakulti Sains, UTM, Skudai

Pemeriksa Dalam II

:

Nama Penyelia Lain (jika ada)

:

Disahkan oleh Penolong Pendaftar di Sekolah Pengajian Siswazah: Tandatangan Nama

: .......................................................... : .GANESAN A/L ANDIMUTHU

Tarikh : ..........................

STRIPPING VOLTAMMETRIC METHODS FOR THE DETERMINATION OF AFLATOXIN COMPOUNDS

MOHAMAD HADZRI BIN YAACOB

A thesis submitted in fulfilment of the requirements for the award of the degree of Doctor of Philosophy

Faculty of Science Universiti Teknologi Malaysia

APRIL 2006

ii

iii

Specially dedicated to:

My mother, wife, sons, daughters and all families for all the love, support and continuous prayer for my success in completing this work.

iv

ACKNOWLEDGEMENT All praise be to ALLAH SWT and blessing be upon His Prophet SAW whose ultimate guidance creates a more meaningful purpose to this work. I wish to express my sincere gratitude and appreciation to the people who have both directly and indirectly contributed to this thesis. The following are those to whom I am particularly indebted: My supervisors A.P. Dr. Abdull Rahim bin Hj. Mohd. Yusoff and Prof. Dr. Rahmalan Ahamad for their invaluable guidance, freedom of work and constant encouragement throughout the course of this work. School Of Health Sciences, USM, Health Campus, Kubang Krian Kelantan for awarding study leave together with scholarship in completing the work. Prof Baharuddin Saad from USM Penang, AP Dr. Razali Ismail from Chemistry Department, Faculty of Science, UTM and Prof Barek from Charles University, Prague, Czeck Republic for their useful discussion and suggestion. Also to Mr Radwan Ismail from Department of Chemistry, Penang Branch, Mrs Marpongahtun Misni and Mr Wan Kamaruzaman Wan Ahmad for their friendship, ideas and continuous support in carrying out this work. Also to Mr Mat Yasin bin Sirin, Mrs Ramlah binti Husin and Mr Azmi Mahmud for their assistance throughout the work. UTM for awarding Short Term Grant No: 75152 / 2004 My mother, wife and all families for their encouragements, supports, patience, tolerance and understanding.

v

ABSTRACT

Aflatoxin, which is produced by Aspergillus flavus and Aspergillus parasiticus fungi is one of the compounds in the mycotoxin group. The main types of aflatoxins are AFB1, AFB2, AFG1 and AFG2 which have carcinogenic properties and are dangerous to human health. Various techniques have been used for their measurements such as the high performance liquid chromatography (HPLC), enzyme linked immunosorbant assay (ELISA) and radioimmunoassay (RIA) but all these methods have disadvantages such as long analysis time, consume a lot of reagents and expensive. To overcome these problems, the voltammetric technique was proposed in this study using controlled growth mercury drop (CGME) as the working electrode and Britton Robinson buffer (BRB) as the supporting electrolyte. The voltammetric methods were used for investigating the electrochemical properties and the quantitative analysis of aflatoxins at the mercury electrode. The experimental conditions were optimised to obtain the best characterised peak in terms of peak height with analytical validation of the methods for each aflatoxin. The proposed methods were applied for the analysis of aflatoxins in groundnut samples and the results were compared with those obtained by the HPLC technique. All aflatoxins were found to adsorb and undergo irreversible reduction reaction at the working mercury electrode. The optimum experimental parameters for the differential pulse cathodic stripping voltammetry (DPCSV) method were the BRB at pH 9.0 as the supporting electrolyte, initial potential (Ei): -0.1 V, final potential (Ef): -1.4 V, accumulation potential (Eacc): 0.6 V, accumulation time (tacc): 80 s, scan rate: 50 mV/s and pulse amplitude: 80 mV. The optimum parameters for the square wave stripping voltammetry (SWSV) method were Ei = -0.1 V, Ef = -1.4 V, Eacc: -0.8 V, tacc: 100 s, scan rate: 3750 mV/s, frequency: 125 Hz and voltage step: 30 V. At the concentration of 0.10 µM, using DPCSV method with the optimum parameters, AFB1, AFB2, AFG1 and AFG2 produced a single peak at -1.21 V, -1.23 V, -1.17 V and -1.15 V (versus Ag/AgCl) respectively. Using the SWSV method, a single peak appeared at -1.30 V for AFB1 and AFB2 while -1.22 V for AFG1 and AFG2. The calibration curves for all aflatoxins were linear with the limit of detection (LOD) of approximately 2.0 ppb and 0.50 ppb obtained by the DPCSV and SWSV methods respectively. The results of aflatoxins content in individual groundnut samples do not vary significantly when compared with those obtained by the HPLC technique. Finally, it can be concluded that both proposed methods which are accurate, precise, robust, rugged, fast and low cost were successfully developed and are potential alternative methods for routine analysis of aflatoxins in groundnut samples.

vi

ABSTRAK

Aflatoksin adalah sejenis sebatian yang dihasilkan oleh kulat Aspergillus flavus dan Aspergillus parasiticus yang digolongkan di dalam kumpulan mikotoksin. Jenis utama aflatoksin adalah AFB1, AFB2, AFG1 dan AFG2 yang bersifat karsinogen serta merbahaya kepada kesihatan manusia. Pelbagai teknik telah digunakan untuk menentukan aflatoksin seperti kromatografi cecair prestasi tinggi (HPLC), asai serapan imuno berikatan enzim (ELISA) dan radioamunoasai (RIA) tetapi teknik-teknik ini mempunyai kelemahan seperti masa analisis yang panjang, melibatkan reagen yang banyak dan kos yang mahal. Untuk mengatasi masalah ini, teknik voltammetri telah dicadangkan untuk kajian aflatoksin menggunakan titisan raksa pembesaran terkawal (CGME) sebagai elektrod bekerja dan larutan penimbal Britton-Robinson (BRB) sebagai elektrolit penyokong. Pelbagai kaedah voltammetri telah digunakan untuk mengkaji sifat elektrokimia aflatoksin pada elektrod raksa dan analisis kuantitatifnya. Parameter kajian telah dioptimumkan untuk memperolehi puncak yang elok berdasarkan ketinggian puncak serta pengesahan analisis untuk kaedah yang dibangunkan bagi setiap aflatoksin. Kaedah ini telah digunakan untuk menentukan kandungan aflatoksin di dalam sampel kacang tanah di mana keputusan yang diperolehi telah dibandingkan dengan keputusan HPLC. Semua aflatoksin yang dikaji didapati terjerap dan menjalani proses tindakbalas penurunan tidak berbalik pada elektrod raksa. Parameter optimum untuk kaedah voltammetri perlucutan kathodik denyut pembeza (DPCSV) adalah larutan BRB pada pH 9.0 sebagai larutan elektrolit, keupayaan awal (Ei): -1.0 V, keupayaan akhir (Ef): -1.4 V, keupayaan pengumpulan (Eacc): -0.6 V, masa pengumpulan (tacc): 80 s, kadar imbasan: 50 mV/s dan amplitud denyut: 80 mV. Untuk kaedah voltammetri perlucutan gelombang bersegi (SWSV), parameter optimum adalah Ei : -1.0 V, Ef : -1.4 V, Eacc: -0.8 V, tacc: 100 s, kadar imbasan: 3750 mV/s, frekuensi: 125 Hz dan beza keupayaan: 30 mV. Menggunakan parameter optimum untuk DPCSV, 0.10 µM AFB1, AFB2, AFG1 dan AFG2 menghasilkan puncak tunggal pada keupayaan -1.21 V, -1.23 V, -1.17 V dan -1.15 V (melawan Ag/AgCl) masingmasingnya. Menggunakan kaedah SWSV, puncak terhasil pada -1.30 V untuk AFB1 dan AFB2, -1.22 V untuk AFG1 dan AFG2. Keluk kalibrasi adalah linear untuk semua aflatoksin dengan had pengesanan (LOD) pada 2.0 dan 0.5 ppb diperolehi dari kaedah DPCSV dan SWSV masing-masingnya. Keputusan analisis kandungan aflatoksin di dalam sampel kacang tanah tidak memberi perbezaan ketara berbanding dengan yang diperolehi menggunakan teknik HPLC. Kesimpulannya, kedua-dua kaedah yang dikaji yang merupakan kaedah yang tepat, jitu, cepat, sesuai digunakan dengan pelbagai model voltammetri dan kos yang murah telah berjaya dibangunkan dan berpotensi besar menjadi kaedah alternatif untuk analisis kandungan aflatoksin di dalam kacang tanah secara berkala.

vii

TABLE OF CONTENTS

CHAPTER

1

TITLE

PAGE

TITLE

i

DECLARATION

ii

DEDICATION

iii

ACKNOWLEDGEMENT

iv

ABSTRACT

v

ABSTRAK

vi

TABLE OF CONTENTS

vii

LIST OF TABLES

xiii

LIST OF FIGURES

xvi

ABBREVATIONS

xxix

LIST OF APPENDICES

xxxiii

LITERATURE REVIEW

1

1.1

Overview

1

1.2

Aflatoxins

3

1.2.1 Aflatoxins in general

3

1.2.2

Chemistry of aflatoxins

5

1.2.3

Health aspects of aflatoxins

12

1.2.4

Analytical methods for the determination of

16

aflatoxins 1.2.5 1.3.

Electrochemical properties of aflatoxins

28

Voltammetric technique

30

1.3.1

30

Voltammetric techniques in general

viii 1.3.2

Voltammetric measurement

31

1.3.2.1 Instrumentation

31

1.3.2.2 Solvent and supporting electrolyte

44

1.3.2.3 Current in voltammetry

46

1.3.2.4 Quantitative and quantitative aspects of

48

voltammetry 1.3.3

Type of voltammetric techniques

49

1.3.3.1 Polarography

49

1.3.3.2 Cyclic voltammetry

51

1.3.3.3 Stripping voltammetry

54

1.3.3.3a Anodic stripping voltammetry

56

1.3.3.3b Cathodic stripping voltammetry

57

1.3.3.3c Adsorptive stripping voltammetry 58 1.3.3.4 Pulse voltammetry

1.4

2

59

1.3.3.4a Differential pulse voltammetry

60

1.3.3.4b Square wave voltammetry

61

Objective and scope of study

64

1.4.1

Objective of study

64

1.4.2

Scope of study

67

RESEARCH METHODOLOGY

70

2.1

Apparatus, material and reagents

70

2.1.1

Apparatus

70

2.1.2

Materials

72

2.1.2.1 Aflatoxin stock and standard solutions

72

2.1.2.2 Real samples

73

Reagents

73

2.1.3.1 Britton Robinson buffer, 0.04 M

73

2.1.3.2 Carbonate buffer, 0.04 M

74

2.1.3.3 Phosphate buffer, 0.04 M

74

2.1.3

ix 2.1.3.4 Ascorbic acid

74

2.1.3.5 β-cyclodextrin solution, 1.0 mM -5

75

2.1.3.6 L-Cysteine, 1.0 x 10 M

75

2.1.3.7 2,4-dihydrofuran, 0.15 M

75

-2

2.1.3.8 Coumarin, 3.0 x 10 M

75

2.1.3.9 Poly-L-lysine, 10 ppm

75

2.1.3.10 Standard aluminium (II) solution, 1.0 mM 75

2.2

2.1.3.11 Standard plumbum(II) solution, 1.0 mM

76

2.1.3.12 Standard zinc (II) solution, 1.0 mM

76

2.1.3.13 Standard copper (II) solution, 1.0 mM

76

2.1.3.14 Standard nickel (II) solution, 1.0 mM

76

2.1.3.15 Methanol: 0.1 N HCl solution, 95%

76

2.1.3.16 Zinc sulphate solution, 15%

76

Analytical Technique

77

2.2.1

General procedure for voltammetric analysis

77

2.2.2

Cyclic voltammetry (Anodic and cathodic

77

directions)

2.2.3

2.2.2.1 Standard addition of sample

77

2.2.2.2 Repetitive cyclic voltammetry

78

2.2.2.3 Effect of scan rate

78

Differential pulse cathodic stripping

78

voltammetric determination of AFB2 2.2.3.1 Effect of pH

79

2.2.3.2 Method optimisation for the determination 79 of AFB2 2.2.3.2a Effect of scan rate

79

2.2.3.2b Effect of accumulation potential

80

2.2.3.2c Effect of accumulation time

80

2.2.3.2d Effect of initial potential

80

2.2.3.2e Effect of pulse amplitude

80

x 2.2.3.3 Method validation

80

2.2.3.4 Interference studies

81

2.2.3.4a Effect of Cu(II), Ni (II), Al(III),

81

Pb(II) and Zn(II) 2.2.3.4b Effect of ascorbic acid,

82

β-cyclodextrin and L-cysteine 2.2.4

2.2.3.5 Modified mercury electrode with PLL

82

Square wave cathodic stripping voltammetry

82

(SWSV)

2.2.5

2.2.4.1 SWSV parameters optimisation

82

2.2.4.2 SWSV determination of all aflatoxins

82

Stability studies of aflatoxins

83

2.2.5.1 Stability of 10 ppm aflatoxins

83

2.2.5.2 Stability of 1 ppm aflatoxins

83

2.2.5.3 Stability of 0.1 µM aflatoxins exposed

83

to ambient temperature 2.2.5.4 Stability of 0.1 µM aflatoxins in different

84

pH of BRB

3

2.2.6 Application to food samples

84

2.2.6.1 Technique 1

84

2.2.6.2 Technique 2

84

2.2.6.3 Technique 3

85

2.2.6.4 Blank measurement

85

2.2.6.5 Recovery studies

85

2.2.6.6 Voltammetric analysis

86

RESULTS AND DISCUSSION

88

3.1

Cyclic voltammetric studies of aflatoxins

88

3.1.1

89

Cathodic and anodic cyclic voltammetric of aflatoxins

xi 3.2

Differential pulse cathodic stripping voltammetry

102

of AFB2 3.2.1 Optimisation of conditions for the stripping

104

analysis 3.2.1.1 Effect of pH and type of supporting

104

electrolyte 3.2.1.2 Optmisation of instrumental conditions

117

3.2.1.2a Effect of scan rate

118

3.2.1.2b Effect of accumulation time

119

3.2.1.2c Effect of accumulation

120

potential

3.2.2

3.2.1.2d Effect of initial potential

121

3.2.1.2e Effect of pulse amplitude

122

Analysis of aflatoxins

127

3.2.2.1 Calibration curves of aflatoxins and

129

validation of the proposed method

3.3

3.2.2.1a Calibration curve of AFB2

129

3.2.2.1b Calibration curve of AFB1

134

3.2.2.1c Calibration curve of AFG1

137

3.2.2.1d Calibration curve of AFG2

140

3.2.2.2 Determination of limit of detection

143

3.2.2.3 Determination of limit of quantification

147

3.2.2.4 Inteference studies

150

Square-wave stripping voltammetry (SWSV) of

157

aflatoxins 3.3.1

SWSV determination of AFB2

158

3.3.1.1 Optimisation of experimental and

159

instrumental SWSV parameters 3.3.3.1a Influence of pH of BRB

159

3.3.3.1b Effect of instrumental

160

variables

xii

3.4

3.3.2 SWSV determination of other aflatoxins

166

3.3.3

168

Calibration curves and method validation

Stability studies of aflatoxins

175

3.4.1

10 ppm aflatoxin stock solutions

175

3.4.2

1 ppm aflatoxins in BRB at pH 9.0

179

3.4.2.1 Month to month stability studies

179

3.4.2.2 Hour to hour stability studies

181

3.4.2.3 Stability studies in different pH

186

of BRB 3.4.2.4 Stability studies in 1.0 M HCl and

191

1.0 M NaOH 3.5

4

Voltammetric analysis of aflatoxins in real samples

192

3.5.1 Study on the extraction techniques

193

3.5.2

Analysis of blank

194

3.5.3

Recovery studies of aflatoxins in real samples

196

3.5.4

Analysis of aflatoxins in real samples

199

CONCLUSIONS AND RECOMMENDATIONS

204

4.1

Conclusions

204

4.2

Recommendations

206

REFERENCES Appendices A – AM

208 255 - 317

xiii LIST OF TABLES

TABLE NO.

TITLE

PAGE

1.0

Scientific name for aflatoxin compounds

7

1.1

Chemical and physical properties of aflatoxin compounds

10

1.2

Summary of analysis methods used for determination of aflatoxins in various samples

19

1.3

Working electrode and limit of detection for modern polarographic and voltammetric techniques.

32

1.4

The application range of various analytical techniques and their concentration limits when compared with the requirements in different fields of chemical analysis

33

1.5

List of different type of working electrodes and its potential windows

36

1.6

Electroreducible and electrooxidisable organic functional groups

50

1.7

The characteristics of different type of electrochemical reaction.

53

1.8

Application of Square Wave Voltammetry technique

62

2.0

List of aflatoxins and their batch numbers used in this experiment

72

2.1

Injected volume of aflatoxins into eluate of groundnut and the final concentrations obtained in voltammetric cell

86

3.0

The dependence of current peaks of aflatoxins to their concentrations obtained by cathodic cyclic measurements in BRB at 9.0.

97

xiv 3.1

Effect of buffer constituents on the peak height of 2.0 µM AFB2 at pH 9.0. Experimental conditions are the same as Figure 3.21

109

3.2

Compounds reduced at the mercury electrode

116

3.3

Optimum parameters for 0.06 µM and 2.0 µM AFB2 in BRB at pH 9.0.

126

3.4

The peak height and peak potential of aflatoxins obtained by optimised parameters in BRB at pH 9.0 using DPCSV technique.

127

3.5

Peak height (in nA) obtained for intra-day and inter-day precision studies of 0.10 µM and 0.20 µM by the proposed voltammetric procedure (n=8).

131

3.6

Mean values for recovery of AFB2 standard solution (n=3).

132

3.7

Influence of small variation in some of the assay condition of the proposed procedure on its suitability and sensitivity using 0.10 µM AFB2.

133

3.8

Results of ruggedness test for proposed method using 0.10 µM AFB2.

134

3.9

Peak height (in nA) obtained for intra-day and inter-day precision studies of 0.10 µM and 0.20 µM AFB1 by proposed voltammetric procedure (n=5).

136

3.10

Mean values for recovery of AFB1 standard solution (n=3).

137

3.11

Peak height (in nA) obtained for intra-day and inter-day precision studies of 0.10 µM and 0.20 µM AFG1 by proposed voltammetric procedure (n=5).

139

3.12

Mean values for recovery of AFG1 standard solution (n=3).

139

3.13

Peak height (in nA) obtained for intra-day and inter-day precision studies of 0.10 µM and 0.20 µM AFG2 by proposed voltammetric procedure.

141

xv 3.14

Mean values for recovery of AFG2 standard solution (n=5).

142

3.15

Peak height and peak potential of 0.10 µM aflatoxins obtained by BAS and Metrohm voltammetry analysers under optimised operational parameters for DPCSV method.

143

3.16

Analytical parameters for calibration curves for AFB1,AFB2, AFG1 and AFG2 obtained by DPCSV technique using BRB at pH 9.0 as the supporting electrolyte.

145

3.17

LOD values for determination of aflatoxins obtained by various methods.

148

3.18

LOQ values for determination of aflatoxins obtained by various methods.

149

3.19

Peak current and peak potential for all aflatoxins obtained by SWSV in BRB at pH 9.0 (n=5).

167

3.20

Analytical parameters for calibration curves for AFB1, AFB2, AFG1 and AFG2 obtained by SWSV technique in BRB pH 9.0 as the supporting electrolyte.

172

3.21

Result of reproducibility study (intra-day and interday measurements) for 0.1 µM aflatoxins in BRB at pH 9.0 obtained by SWSV method.

173

3.22

Application of the proposed method in evaluation of the SWSV method by spiking the aflatoxin standard solutions.

174

3.23

Average concentration of all aflatoxins within a year stability studies.

175

3.24

The peak current and peak potential of 10 ppb AFB2 in presence and absence of a blank sample.

196

3.25

Total aflatoxin contents in real samples which were obtained by DPCSV and HPLC techniques (average of duplicate analysis)

203

xvi LIST OF FIGURES

FIGURE NO.

TITLE

PAGE

1.0

Aspergillus flavus seen under an electron microscope.

4

1.1

Chemical structure of coumarin

6

1.2

Chemical structures of (a) AFB1, (b) AFB2, (c) AFG1, (d) AFG2, (e) AFM1 and (f) AFM2

8

1.3

Hydration of (a) AFB1 and (b) AFG1 by TFA produces (c) AFB2a and (d) AFG2a

9

1.4

Transformation of toxic (a) AFB1 to non-toxic (b) aflatoxicol A.

11

1.5

Major DNA adducts of AFB1; (a) 8,9-Dihydro-8(N7-guanyl)-9-hydroxy-aflatoxin B1 (AFB1-Gua) and (b) 8,9-Dihydro-8-(N5-Formyl-2’.5’.6’triamino-4’-oxo-N5-pyrimidyl)-9-hydroxy-Aflatoxin B1 (AFB1-triamino-Py)

13

1.6

Metabolic pathways of AFB1 by cytochrom P-450 enzymes; B1-epoxide = AFB1 epoxide, M1= aflatoxin M1, P1= aflatoxin P1 and Q1 = aflatoxin Q1.

14

1.7

A typical arrangement for a voltammetric electrochemical cell (RE: reference electrode, WE: working electrode. AE: auxiliary electrode)

34

1.8

A diagram of the Hanging Mercury Drop Electrode (HMDE)

37

1.9

A diagram of the Controlled Growth Mercury Electrode (CGME)

38

1.10

Cyclic voltammograms of (a) reversible, (b) irriversible and (c) quasireversible reaction at mercury electrode (O = oxidised form and R = reduced form)

52

xvii

1.11

The potential-time sequence in stripping analysis

55

1.12

Schematic drawing showing the Faradaic current and charging current versus pulse time course

59

1.13

Schematic drawing of steps in DPV by superimposing a periodic pulse on a linear scan

60

1.14

Waveform for square-wave voltammetry

61

2.0

BAS CGME stand (a) which is connected to CV-50W voltammetric analyser and interface with computer (b) for data processing

71

2.1

VA757 Computrace Metrohm voltammetric analyser with 663 VA stand (consists of Multi Mode (MME))

71

3.0

Cathodic peak current of 0.6 µM AFB1 in various pH of BRB obtained in cathodic cyclic voltammetry. Ei = 0, Elow = -1.5 V, Ehigh = 0 and scan rate = 200 mV/s)

89

3.1

Shifting of peak potential of AFB1 with increasing pH of BRB. Parameter conditions are the same as in Figure 3.0.

90

3.2

Mechanism of reduction of AFB1 in BRB at pH 6.0 to 8.0.

90

3.3

Mechanism of reduction of AFB1 in BRB at pH 9.0 to 11.0.

91

3.4

Cathodic cyclic voltammogram for 1.3 µM AFB1 obtained at scan rate of 200 mV/s, Ei = 0, Elow = -1.5 V and Ehigh = 0 in BRB solution at pH 9.0.

92

3.5

Cathodic cyclic voltammogram for 1.3 µM AFB2 in BRB solution at pH 9.0. All parameter conditions are the same as in Figure 3.4.

92

3.6

Cathodic cyclic voltammogram for 1.3 µM AFG1 in BRB solution at pH 9.0. All parameter conditions are the same as in Figure 3.4.

93

xviii 3.7

Cathodic cyclic voltammogram for 1.3 µM AFG2 in BRB solution at pH 9.0. All parameter conditions are the same as in Figure 3.4.

93

3.8

Effect of Ei to the Ip of 0.6 µM AFB1 in BRB at pH 9.0 obtained by cathodic cyclic voltammetry. All parameter conditions are the same as in Figure 3.4.

94

3.9

Effect of Ei to the Ip of 0.1 µM Zn2+ in BRB at pH 9.0 obtained by cathodic cyclic voltammetry. All parameter conditions are the same as in Figure 3.4.

94

3.10

Anodic cyclic voltammogram of 1.3 µM AFB2 obtained at scan rate of 200 mV/s, Ei = -1.5 V, Elow = -1.5 V and Ehigh = 0 in BRB at pH 9.0.

95

3.11

Effect of increasing AFB2 concentration on the peak height of cathodic cyclic voltammetrc curve in BRB at pH = 9.0. (1.30 µM, 2.0 µM, 2.70 µM and 3.40 µM). All parameter conditions are the same as in Figure 3.4.

96

3.12

Peak height of reduction peak of AFB2 with increasing concentration of AFB2. All parameter conditions are the same as in Figure 3.4.

96

3.13

Repetitive cathodic cyclic voltammograms of 1.3 µM AFB2 in BRB solution at pH 9.0. All experimental conditions are the same as in Figure 3.4.

98

3.14

Increasing Ip of 1.3 µM AFB2 cathodic peak obtained from repetitive cyclic voltammetry. All experimental conditions are the same as in Figure 3.4.

98

3.15

Peak potential of 1.3 µM AFB2 with increasing number of cycle obtained by repetitive cyclic voltammetry.

99

xix 3.16

Plot of log Ip versus log υ for 1.3 µM AFB2 in BRB solution at pH 9.0. All experimental conditions are the same as in Figure 3.4.

100

3.17

Plot of Ep versus log υ for 1.3 µM AFB2 in BRB solution at pH 9.0.

101

3.18

Plot of Ip versus υ for 1.3 µM AFB2 in BRB solution at pH 9.0. All parameter conditions are the same as in Figure 3.4.

102

3.19

DPCS voltammograms of 1.0 µM AFB2 (Peak I) in BRB at pH 9.0 (a) at tacc = 0 and 30 s. Other parameter conditions; Ei = 0, Ef = -1.50 V, Eacc = 0, υ =50 mV/s and pulse amplitude = 100 mV. Peak II is the Zn peak.

103

3.20

DPCS voltammograms of 2.0 µM AFB2 in BRB in BRB (Peak I) at different pH values; 6.0, 7.0, 8.0, 9.0, 11.0and 13.0. Other parameter conditions; Ei = 0, Ef =-1.50 V, Eacc = 0, υ = 50 mV/s and pulse amplitude =100 mV. Peak II is the Zn peak.

104

3.21

Dependence of the Ip for AFB2 on the pH of 0.04 M BRB solution. AFB2 concentration: 2.0 µM, Ei =0, Ef = -1.5 V, E acc = 0, tacc = 30 sec, υ = 50 mV/s and pulse amplitude = 100 mV.

105

3.22

Ip of 2.0 µM AFB2 obtained in BRB (a) at pH from 9.0 decreases to 4.0 and re-increase to 9.0 and (b) at pH from 9.0 increase to 13.0 and re-decrease to 9.0.

106

3.23

UV-VIS spectrums of 1 ppm AFB2 in BRB at pH (a) 6.0, (b) 9.0 and (c) 13.0.

107

3.24

Opening of lactone ring by strong alkali caused no peak to be observed for AFB2 in BRB at pH 13.0.

108

3.25

Ip of 2.0 µM AFB2 in different concentration of BRB at pH 9.0. Experimental conditions are the same as in Figure 3.20.

109

3.26

Ip of 2.0 µM AFB2 in different pH and concentrations of BRB.

110

xx

3.27

DPCS voltammograms of 2.0 AFB2 (Peak I) in (a) 0.04 M, (b) 0.08 M and (c) 0.08 M BRB at pH 9.0 as the blank. Ei = 0 V, Ef = -1.5 V, Eacc = 0 V, tacc = 30 sec, υ = 50mV/s and pulse amplitude = 100 mV. Peak II is the Zn peak.

110

3.28

Chemical structures of (a) 2,3-dihydrofuran, (b) tetrahydrofuran and (c) coumarin.

111

3.29

Voltammograms of 2,3-dihydrifuran (peak I) for concentrations from (b) 0.02 to 0.2 µM in (a) BRB at pH 9.0.

112

3.30

Dependence of Ip of coumarin to its concentrations

112

3.31

Voltammograms of coumarin at concentration of (b) 34 µM, (c) 68 µM, (d) 102 µM, (e) 136 µM and (f) 170 µM in (a) BRB at pH 9.0.

113

3.32

Chemical structures of (a) cortisone and (b) testosterone.

114

3.33

Chemical structures of (a) digoxin and (b) digitoxin

115

3.34

Effect of pH of BRB solution on the Ep for AFB2. AFB2 concentration: 2.0 µM. Ei = 0, Ef = -1.5 V, Eacc = 0, t acc = 30 s and υ = 50 mV/s.

117

3.35

Effect of various υ to the (a) Ip and (b) Ep of 2.0 µM AFB2 peak in BRB at pH 9.0. Ei = 0, Ef = 1.50 V, Eacc = 0, t acc = 15 s and pulse amplitude = 100 mV.

118

3.36

Effect of tacc on (a) Ip and (b) Ep of 2.0 µM AFB2 peak in BRB at pH 9.0. Ei = 0, Ef = 1.50 V, Eacc = 0, υ = 40 mV/s and pulse amplitude = 100 mV.

119

3.37

The relationship between (a) Ip and (b) Ep with Eacc for 2.0 µM AFB2 in BRB at pH 9.0. Ei = 0, Ef = -1.5 V, tacc = 15 s, υ = 40 mV/s and pulse amplitude = 100 mV.

120

3.38

Effect of Ei on (a) Ip and (b) Ep of 2.0 µM AFB2 in BRB at pH 9.0. Ef = 1.50 V, Eacc = -0.80 V, tacc = 40 s,υ = 40 mV/s and pulse amplitude = 100 mV.

121

xxi 3.39

Effect of pulse amplitude on (a) Ip and (b) Ep of 2.0 µM AFB2 in BRB at pH 9.0. Ei = -1.0 V, Ef = 1.50 V, Eacc = -0.80 V, tacc = 40 s and υ = 40 mV/s.

122

3.40

Effect of Eacc on Ip of 0.06 µM AFB2. Ei = -1.0 V, Ef = -1.50 V, tacc = 40 s, υ = 40 mV/s and pulse amplitude = 100 mV.

123

3.41

Effect of tacc on (a) Ip and (b) Ep of 0.06 µM AFB2 in BRB at pH 9.0. Ei = -1.0 V, Ef = -1.50 V, Eacc = -0.6 V, υ = 40 mV/s and pulse amplitude = 100 mV.

124

3.42

The effect υ on (a) Ip and (b) Ep of 0.06 µM AFB2 In BRB at pH 9.0. Ei = -1.0 V, E f = -1.50 V, Eacc = -0.6 V, tacc = 80 s and pulse amplitude = 100 mV.

125

3.43

Voltammograms of 0.06 µM AFB2 obtained under (a) optimised and (b) unoptimised parameters in BRB at pH 9.0.

126

3.44

Voltammograms of 0.1 µM (a) AFB1, (b) AFG1, (c) AFB2 and (d) AFG2 in BRB at pH 9.0. Ei = 1.0 (except for AFG1 = -0.95 V), Ef = -1.4 V, Eacc = -0.6 V, tacc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV.

128

3.45

Voltammograms of (b) mixed aflatoxins in (a) BRB at pH 9.0 as the blank. Parameters condition: Ei = -0.95 V, Ef = -1.4 V, Eacc = -0.6 V, t acc = 80 s, υ = 50 mV/s and Pulse amplitude = 80 mV.

129

3.46

Increasing concentration of AFB2 in BRB at pH 9.0. The parameter conditions: Ei = -1.0 V, Ef = -1.4 V, Eacc =-0.6 V, t acc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV.

130

3.47

Linear plot of Ip versus concentration of AFB2 in BRB at pH 9.0. The parameter conditions are the same as in Figure 3.46.

130

3.48

Standard addition of AFB1 in BRB at pH 9.0. The parameter conditions: Ei = -1.0 V, Ef = -1.4 V, Eacc =-0.8 V, t acc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV.

135

xxii 3.49

Linear plot of Ip versus concentration of AFB1 in BRB at pH 9.0. The parameter conditions are the same as in Figure 3.48.

135

3.50

Effect of concentration to Ip of AFG1 in BRB at pH 9.0. Ei = -0.95 V, Ef = -1.40 V, Eacc =-0.8 V, tacc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV.

138

3.51

Linear plot of Ip versus concentration of AFG1 in BRB at pH 9.0. The parameter conditions are the same as in Figure 3.50.

138

3.52

Effect of concentration to Ip of AFG2 in BRB at pH 9.0. Ei = -1.0 V, Ef = -1.40 V, Eacc =-0.8 V, tacc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV.

140

3.53

Linear plot of Ip versus concentration of AFG2 in BRB at pH 9.0. The parameter conditions are the same as in Figure 3.52.

141

3.54

Voltammograms of 0.1 µM (a) AFB1, (b) AFB2, (c) AFG1 and (d) AFG2 in BRB at pH 9.0 (d) obtained by 747 VA Metrohm. Ei = -1.0 V (except for AFG1 = -0.95 V), Ef = -1.4 V, Eacc = -0.6 V, tacc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV.

144

3.55

Ip of all aflatoxins with increasing concentration of Zn 2+ up to 1.0 µM.

150

3.56

Voltammograms of (i) 0.1µM AFB2 and (ii) AFB2-Zn 151 complex with increasing concentration of Zn2+ (a = 0, b = 0.75 µM, c = 1.50 µM, d = 2.25 µM and e = 3.0 µM). Blank = BRB at pH 9.0. Experimental conditions; Ei = -1.0 V, Ef = -1.40 V, Eacc = -0.6 V, tacc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV.

3.57

Ip of all aflatoxins after reacting with increasing concentration of Zn2+ in BRB at pH 3.0. Measurements were made in BRB at pH 9.0 within 15 minutes of reaction time. Ei = -1.0 V (except for AFG1 = -0.95 V), Ef = -1.40 V, Eacc = -0.6 V, tacc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV.

151

xxiii 3.58

Absorbance of all aflatoxins with increasing concentration of Zn2+ in BRB at pH 3.0 within 15 minutes of reaction time

152

3.59

Voltammograms of 0.1µM AFB2 with increasing concentration of Zn2+ (from 0.10 to 0.50 µM) in BRB at pH 9.0. Ei = -0.25 V, Ef = -1.4 V, Eacc = -0.6 V, tacc = 80 s, υ = 50 mV/s and pulse amplirude = 80 mV. .

153

3.60

Chemical structure of ascorbic acid.

153

3.61

Ip of all aflatoxins with increasing concentration of ascorbic acid up to 1.0 µM. Concentrations of all aflatoxins are 0.1µM.

154

3.62

Voltammograms of 0.1µM AFB2 with increasing concentration of ascorbic acid.

154

3.63

Ip of all aflatoxins with increasing concentration of β-cyclodextrin up to 1.0 µM.

155

3.64

Voltammograms of 0.1µM AFB2 with increasing concentration of β-cyclodextrin.

156

3.65

Chemical structure of L-cysteine.

156

3.66

Ip of all aflatoxins with increasing concentration of cysteine up to 1.0 µM.

157

3.67

Voltammograms of 0.1 µM AFB2 obtained by (a) DPCSV and (b) SWSV techniques in BRB at pH 9.0. Parameters for DPCSV: Ei = -1.0 V, Ef = -1.40V, Eacc = -0.6 V, t acc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV and for SWSV: Ei = -1.0 V, Ef = -1.40V, Eacc = -0.6 V, tacc = 80 s, frequency = 50 Hz, voltage step = 0.02 V, amplitude = 80 mV and υ = 1000 mV/s.

158

3.68

Influence of pH of BRB on the Ip of 0.10 µM AFB2 using SWSV technique. The instrumental Parameters are the same as in Figure 3.67.

159

3.69

Voltammograms of 0.1 µM AFB2 in different pH of BRB. Parameter conditions: Ei = -1.0 V, E f = -1.40 V, Eacc = -0.6 V, tacc = 80 s, voltage step =

160

xxiv 0.02 V, amplitude = 50 mV, frequency = 50 Hz and υ = 1000 mV/s. 3.70

Effect of Ei to the Ip of 0.10 µM AFB2 in BRB at pH 9.0.

160

3.71

Effect of Eaccto the Ip of 0.10 µM AFB2 in BRB at pH 9.0.

161

3.72

Relationship between Ip of 0.10 µM AFB2 while increasing tacc.

162

3.73

Effect of frequency to the Ip of 0.10 µM AFB2 in BRB at pH 9.0.

162

3.74

Linear relatioship between Ip of AFB2 and square root of frequency.

163

3.75

Influence of square-wave voltage step to Ip of 0.10 µM AFB2.

163

3.76

Influence of square-wave amplitude to Ip of 0.10 µM AFB2.

164

3.77

Relationship of SWSV Ep of AFB2 with increasing amplitude.

164

3.78

Ip of 0.10 µM AFB2 obtained under (a) non-optimised and (b) optimised SWSV parameters compared with that obtained under (c) optimised DPCSV parameters.

165

3.79

Voltammograms of 0.10 µM AFB2 obtained under (a) non-optimised and (b) optimised SWSV parameters compared with that obtained under (c) optimised DPCSV parameters.

166

3.80

Ip of 0.10 µM aflatoxins obtained using two different stripping voltammetric techniques under their optimum paramter conditions in BRB at pH 9.0.

167

3.81

Voltammograms of (i) AFB1, (ii) AFB2, (iii) AFG1 and (iV) AFG2 obtained by (b) DPCSV compared with that obtained by (c) SWSV in (a) BRB at pH 9.0.

168

3.82

SWSV voltammograms for different concentrations of AFB1 in BRB at pH 9.0. The broken line

169

xxv represents the blank: (a) 0.01 µM, (b) 0.025 µM, (c) 0.05 µM, (d) 0.075 µM, (e) 0.10 µM, (f) 0.125 µM and (g) 0.150 µM. Parameter conditions: Ei = -1.0 V, Ef = -1.4 V, Eacc = -0.8 V, t acc = 100 s, frequency = 125 Hz, voltage step = 0.03 V, pulse amplitude = 75 mV and scan rate = 3750 mV/s. 3.83

Calibration curve for AFB1 obtained by SWSV method.

170

3.84

Calibration curve for AFB2 obtained by SWSV method.

170

3.85

Calibration curve for AFG1 obtained by SWSV method.

170

3.86

Calibration curve for AFG2 obtained by SWSV method.

171

3.87

LOD for determination of aflatoxins obtained by two different stripping methods.

171

3.88

UV-VIS spectrums of 10 ppm of all aflatoxins in benzene: acetonitrile (98%) at preparation date.

176

3.89

UV-VIS spectrums of 10 ppm of all aflatoxins in benzene: acetonitrile (98%) after 6 months of storage time.

176

3.90

UV-VIS spectrums of 10 ppm of all aflatoxins in benzene: acetonitrile (98%) after 12 months of storage time.

177

3.91

UV-VIS spectrums of 10 ppm AFB1 (a) kept in the cool and dark conditions and (b) exposed to ambient conditions for 3 days.

178

3.92

UV-VIS spectrums of 1 ppm AFB1 in BRB solution prepared from (a) good and (b) damaged 10 ppm AFB1 stock solution.

178

3.93

UV-VIS spectrums of 1 ppm AFB1 in BRB solution prepared from (a) good and (b) damaged 10 ppm AFB1 stock solution after 2 week stored in the cool and dark conditions.

179

xxvi 3.94

Percentage of Ip of 0.10 µM aflatoxins in BRB at pH 9.0 at different storage time in the cool and dark conditions.

180

3.95

Percentage of Ip of all aflatoxins in BRB at pH 9.0 exposed to ambient conditions up to 8 hours of exposure time.

181

3.96

Reaction of 8,9 double bond furan rings in AFB1 with TFA, iodine and bromine under special conditions (Kok, et al., 1986).

182

3.97

Voltammograms of 0.10 µM AFB1 obtained in (a) BRB at pH 9.0 which were prepared from (b) damaged and (c) fresh stock solutions.

183

3.98

Ip of 0.10 µM AFB1 obtained in BRB at pH 9.0 from 0 to 8 hours in (a) light exposed and (b) light protected.

184

3.99

Ip of 0.10 µM AFB2 obtained in BRB at pH 9.0 from 0 to 8 hours in (a) light exposed and (b) light protected.

184

3.100

Ip of 0.10 µM AFG1 obtained in BRB at pH 9.0 from 0 to 8 hours in (a) light exposed and (b) light protected.

185

3.101

Ip of 0.10 µM AFG2 obtained in BRB at pH 9.0 from 0 to 8 hours in (a) light exposed and (b) light protected.

185

3.102

Peak heights of 0.10 µM aflatoxins in BRB at pH (a) 6.0, (b) 7.0, (c) 9.0 and (d) 11.0 exposed to ambient conditions up to 3 hours of exposure time.

187

3.103

Resonance forms of the phenolate ion (Heathcote, 1984).

187

3.104

Voltammograms of 0.10 µM AFB2 in BRB at pH 6.0 from 0 to 3 hrs exposure time.

188

3.105

Voltammograms of 0.10 µM AFB2 in BRB at pH 11.0 from 0 to 3 hrs of exposure time.

188

xxvii 3.106

Voltammograms of 0.10 µM AFG2 in BRB at pH 6.0 from 0 to 3 hrs of exposure time.

189

3.107

Voltammograms of 0.10 µM AFG2 in BRB at pH 11.0 from 0 to 3 hrs of exposure time.

189

3.108

Absorbance of 1.0 ppm aflatoxins in BRB at pH (a) 6.0, (b) 7.0, (c) 9.0 and (d) 11.0 from 0 to 3 hours of exposure time.

190

3.109

The peak heights of aflatoxins in 1.0 M HCl from 0 to 6 hours of reaction time.

191

3.110

The peak heights of aflatoxins in 1.0 M NaOH from 0 to 6 hours of reaction time.

192

3.111

Voltammograms of real samples after extraction by Technique (a) 1, (b) 2 and (c) 3 with addition of AFB1 standard solution in BRB at pH 9.0 as a blank.

193

3.112

Voltammograms of blank in BRB at pH 9.0 obtained by (a) DPCSV and (b) SWSV methods.

194

3.113

DPCSV (a) and SWSV (b) voltammograms of 10 ppb AFB2 (i) in present of blank sample (ii) obtained in BRB at pH 9.0 (iii) as the supporting electrolyte.

195

3.114

DPCSV voltammograms of real samples (b) added with 3 ppb (i), 9 ppb (ii) and 15 ppb (iii) AFB1 obtained in BRB at pH 9.0 (a) as the blank on the first day measurement.

197

3.115

Percentage of recoveries of (a) 3 ppb, (b) 9 ppb, (c) 15 ppb of all aflatoxins in real samples obtained by DPCSV methods for one to three days of measurements.

198

3.116

DPCSV voltammograms of real sample, S11 (b) with the addition of 10 ppb AFB1 (c) in BRB at pH 9.0 (a) as the blank. Parameter conditions: Eacc = -0.6 V, tacc = 80 s, scan rate = 50 mV/s and pulse amplitude = 80 mV.

199

xxviii 3.117

SWSV voltammograms of real sample, S11 (b) with the addition of 10 ppb AFB1 (c) in BRB at pH 9.0 (a) as the blank. Parameter conditions: Eacc = -0.8 V, tacc = 100 s, scan rate = 3750 mV/s, frequency = 125 Hz, voltage step = 0.03 V and pulse amplitude = 75 mV.

200

3.118

DPCSV voltammograms of real sample, S07 (b) with the addition of 10 ppb AFB1 (c) in BRB at pH 9.0 (a) as the blank. Parameter conditions: Eacc = -0.6 V, tacc = 80 s, scan rate = 50 mV/s and pulse amplitude = 80 mV.

200

3.119

SWSV voltammograms of real sample, S07 (b) with the addition of 10 ppb AFB1 (c) in BRB at pH 9.0 (a) as the blank. Parameter conditions: Eacc = -0.8 V, tacc = 100 s, scan rate = 3750 mV/s, frequency = 125 Hz, voltage step = 0.03 V and pulse amplitude = 75 mV.

201

3.120

DPCSV voltammograms of real sample, S10 (b) with the addition of 10 ppb AFB1 (c) in BRB at pH 9.0 (a) as the blank. Parameter conditions: Eacc = -0.6 V, tacc = 80 s, scan rate = 50 mV/s and pulse amplitude = 80 mV.

201

3.121

SWSV voltammograms of real sample, S10 (b) with the addition of 10 ppb AFB1 (c) in BRB at pH 9.0 (a) as the blank. Parameter conditions: Eacc = -0.8 V, tacc = 100 s, scan rate = 3750 mV/s, frequency = 125 Hz, voltage step = 0.03 V and pulse amplitude = 80 mV.

202

xxix ABBREVIATIONS

AAS

Atomic absorption spectrometry

Abs

Absorbance

ACP

Alternate current polarography

ACV

Alternate current voltammetry

AD

Amperometric detector

AdCSV

Adsorptive cathodic stripping voltammetry

AE

Auxiliary electrode

AFB1

Aflatoxin B1

AFB2

Aflatoxin B2

AFG1

Aflatoxin G1

AFG2

Aflatoxin G2

AFM1

Aflatoxin M1

AFM2

Aflatoxin M2

AFP1

Aflatoxin P1

AFQ1

Aflatoxin Q1

Ag/AgCl

Silver/silver chloride

ASV

Anodic stripping voltammetry

β-CD

β-cyclodextrin

BFE

Bismuth film electrode

BLMs

Bilayer lipid membranes

BRB

Britton Robinson Buffer

CA

Concentration of analyte

CE

Capillary electrophoresis

CGME

Controlled growth mercury electrode

CME

Chemically modified electrode

CPE

Carbon paste electrode

CSV

Cathodic stripping voltammetry

CV

Cyclic voltammetry

xxx DC

Direct current

DCP

Direct current polarography

DME

Dropping mercury electrode

DMSO

Dimethyl sulphonic acid

DNA

Deoxyribonucliec acid

DPCSV

Differential pulse cathodic stripping voltammetry

DPP

Differential pulse polarography

DPV

Differential pulse voltammetry

Eacc

Accumulation potential

Ei

Initial potential

Ef

Final potential

Ehigh

High potential

Elow

Low potential

Ep

Peak potential

ECS

Electrochemical sensing

Et4NH4 OH

Tetraethyl ammonium hydroxide

ELISA

Enzyme linked immunosorbant assay

FD

Fluorescence detector

FDA

Food and Drug Administration

GCE

Glassy carbon electrode

GC-FID

Gas chromatography with flame ionisation detector

HMDE

Hanging mercury drop electrode

HPLC

High performance liquid chromatography

HPTLC

High pressure thin liquid chromatography

IAC

Immunoaffinity chromatography

IACLC

Immunoaffinity column liquid chromatography

IAFB

Immunoaffinity fluorometer biosensor

IARC

International Agency for Research Cancer

Ic

Charging current

Id

Diffusion current

If

Faradaic current

xxxi Ip

Peak height

ICP-MS

Induced coupled plasma-mass spectrometer

IR

Infra red

IUPAC

International Union of Pure and Applied Chemistry

KGy

Kilogray

LD50

Lethal dose 50

LOD

Limit of detection

LOQ

Limit of quantification

LSV

Linear sweep voltammetry

MFE

Mercury film electrode

MS

Mass spectrometer

MECC

Micellar electrokinetic capillary chromatography

MOPS

3-(N-morpholino)propanesulphonic

MOSTI

Ministry of Science, Technology and Innovation

NP

Normal polarography

NPP

Normal pulse polarography

NPV

Normal pulse voltammetry

OPLC

Over pressured liquid chromatography

PAH

Polycyclic aromatic hydrocarbon

PLL

Poly-L-lysine

ppb

part per billion

ppm

part per million

PSA

Potentiometric stripping analysis

RDX

Hexahydro-1,3,5-trinitro-1,3,5-triazine

RE

Reference electrode

RIA

Radioimmunoassay

RNA

Ribonucleic acid

RSD

Relative standard deviation

S/N

Signal to noise ratio

SCE

Standard calomel electrode

SCV

Stair case voltammetry

xxxii SDS

Sodium dodecyl sulphate

SHE

Standard hydrogen electrode

SIIA

Sequential injection immunoassay

SMDE

Static mercury drop electrode

SPE

Solid phase extraction

SPCE

Screen printed carbon electrode

SWP

Square-wave polarography

SWV

Square-wave voltammetry

SWSV

Square-wave stripping voltammetry

SV

Stripping voltammetry

tacc

Accumulation time

TBS

Tris buffered saline

TEA

Triethylammonium

TLC

Thin layer chromatography

TFA

Trifluoroacetic acid

UME

Ultra microelectrode

ν

Scan rate

v/v

Volume per volume

UVD

Ultraviolet-Visible detector

UV-VIS

Ultraviolet-Visible

WE

Working electrode

WHO

World Health Organisation

λmax

Maximum wavelenght

εmax

Maximum molar absorptivity

xxxiii LIST OF APPENDICES

APPENDIX

TITLE

PAGE

A

Relative fluorescence of aflatoxins in different solvent s

255

B

UV spectra of the principal aflatoxins (in methanol)

256

C

Relative intensities of principal bands in the IR spectra of the aflatoxins

257

D

Calculation of concentration of aflatoxin stock solution

258

E

Extraction procedure for aflatoxins in real samples

259

F

Calculation of individual aflatoxin in groundnut samples.

260

G

Cyclic voltammograms of AFB1, AFB2 and AFG2 with increasing of their concentrations.

262

H

Dependence if the peak heights of AFB1, AFG1 and AFG2 on their concentrations.

264

I

Repetitive cyclic voltammograms and their peak heights of AFB1, AFG1 and AFG2 in BRB at pH 9.0

266

J

Plot Ep – log scan rate for the reduction of AFB1, AFG1 and AFG2 in BRB at pH 9.0

270

K

Plot of peak height versus scan rate for 1.3 µM of AFB1, AFG1 and AFG2 in BRB at pH 9.0

272

L

Voltammograms of AFB2 with increasing concentration.

274

M

Voltammograms of 0.1 µM and 0.2 µM AFB2 obtained on the same day measurements

275

N

Voltammograms of AFB2 at inter-day measurements

276

xxxiv

O

F test for robustness and ruggedness tests

278

P

Voltammograms of AFB1 with increasing concentration

280

Q

Voltammograms of AFG1 with increasing concentration

281

R

Voltammograms of AFG2 with increasing concentration

282

S

LOD determination according to Barek et al. (2001a)

283

T

LOD determination according to Barek et al. (1999)

286

U

LOD determination according to Zhang et al. (1996)

287

V

LOD determination according to Miller and Miller (1993)

288

W

ANOVA test

290

X

Peak height of aflatoxins in presence and absence of PLL

292

Y

SWSV voltammograms of AFB1, AFB2, AFG1 and AFG2 in BRB at pH 9.0

293

Z

SWSV voltammograms of 0.10 µM AFB1, AFB2, AFG1 and AFG2 in BRB at pH 9.0

295

AA

UV-VIS spectrums of 10 ppm AFB1, AFB2, AFG1 and AFG2 stock solutions

297

AB

Voltammograms of AFB1, AFB2, AFG1 and AFG2 obtained from 0 to 6 months of storage time in the cool and dark conditions.

299

AC

Voltammograms of AFB1, AFB2, AFG1 and AFG2 in BRB at pH 9.0 from 0 to 8 hours of exposure time.

302

AD

UV-VIS spectrums of AFB2 in BRB at pH 6.0 and 11.0.

305

xxxv AE

Voltammograms of AFB1 and AFG1 in 1.0 M HCl and 1.0 M NaOH

307

AF

DPCSV voltammograms of real samples added with various concentrations of AFG1

309

AG

SWSV voltammograms of real samples added with various concentrations of AFB1.

310

AH

Percentage of recoveries of various concentrations of all aflatoxins (3.0 and 9.0 ppb) in real samples obtained by SWSV method.

311

AI

Calculation of percentage of recovery for 3.0 ppb AFG1 added into real samples.

312

AJ

HPLC chromatograms of real samples: S10 and S07

313

AK

Calculation of aflatoxin in real sample, S13

314

AL

List of papers presented or published to date resulting from this study.

315

AM

ICP-MS results for analysis of BRB at pH 9.0

317

CHAPTER I

LITERATURE REVIEW

1.1

Overview Humans are continuously exposed to varying amounts of chemicals that have

been shown to have carcinogenic or mutagenic properties in environmental systems. Exposure can occur exogenously when these agents are present in food, air or water, and also endogenously when they are products of metabolism or pathophysiologic states such as inflammation. Great attention is focused on environmental health in the past two decades as a consequence of the increasing awareness over the quality of life due to major environment pollutants that affect it. Studies have shown that exposure to environmental chemical carcinogens have contributed significantly to cause human cancers, when exposures are related to life style factors such as diet (Wogan et al., 2004). The contamination of food is part of the global problem of environmental pollution. Foodstuffs have been found contaminated with substances having carcinogenic, mutagenic, teratogenic and allergenic properties. As these substances can be supplied with food throughout the entire life-time of a person, it is necessary to deal with the chronic action of trace amounts of such substances. Hence the systematic determination of the foreign substances in nutritional products and feedstock plays an important role. The determination of trace impurities presents considerable difficulties owing to the fact that food is a complex system containing thousands of major and minor compounds (Nilufer and Boyacio, 2002). Increasing environment pollution by toxic substances such as toxic metals, organometallic and organic pollutants in air,

2 water, soil and food, calls for reliable analytical procedures for their control in environmental samples which needs reliable and sensitive methods (Fifield and Haines, 2000). The choice of the method of analysis depends on the sample, the analyte to be assayed, accuracy, limit of detection, cost and time to complete the analysis (AboulEneim et a., 2000). For development of this method, emphasis should be on development of simplified, cost-effective and efficient method that complies with the legislative requirements (Stroka and Anklam, 2002; Enker, 2003). The widespread occurrence of aflatoxins producing fungi in our environment and the reported naturally occurring of toxin in a number of agricultural commodities has led the investigator to develop a new method for aflatoxin analysis (Creepy, 2002). An accurate and sensitive method of analysis is therefore required for the determination of these compounds in foodstuffs that have sustained mould growth. Numerous articles concerning methods for determination of aflatoxins have been published. However, with regard to electroanalytical technique, only one method of determination was reported using the differential pulse pulse polarographic (DPP) technique which was developed by Smyth et al. (1979). In this experiment, the obtained limit of detection of aflatoxin B1 was 25 ppb which was higher as compared to the common amount of aflatoxin in contaminated food samples which is 10 ppb as reported by Pare (1997) or even less. In Malaysia, the regulatory limit for total aflatoxins in groundnut is 15 ppb. The regulatory for other foods and milk is 10 ppb and 0.05 ppb respectively (Malaysian Food Act, 1983).

1.2

AFLATOXINS

1.2.1 Aflatoxins in General Aflatoxins are a group of heterocyclic, oxygen-containing mycotoxins that possess the bisdifuran ring system. It was discovered some 43 years ago in England

3 following a poisoning outbreak causing 100,000 turkeys death (Miller, 1987 and Cespedez and Diaz, 1997). The aflatoxins are the most widely distributed fungal toxins in food. The occurrence of the aflatoxins in food products demonstrated that the high levels of aflatoxins are significant concern both for food traders and food consumers (Tozzi et al. 2003; Herrman, 2004; Haberneh, 2004). Aflatoxin is a by-product of mold growth in a wide range of agriculture commodities such as peanuts (Urano et al., 1993), maize and maize based food (Papp et al., 2002; Mendez-Albores et al., 2004), cottonseeds (Pons and Franz, 1977), cocoa (Jefferey et al., 1982), coffee beans (Batista et al., 2003), medical herbs ( Reif and Metzger, 1998; Rizzo et al., 2004), spices (Erdogen, 2004; Garner et al., 1993; Akiyama et al., 2001; Aziz et al., 1998), melon seeds (Bankole et al., 2004) and also in human food such as rice (Shotwell et al., 1966; Begum and Samajpati, 2000), groundnut (Bankole et al., 2005), peanut products ( Patey et al., 1990), corn ( Shotwell and Goulden, 1977; Urano et al., 1993 ), vegetable oil (Miller et al., 1985), beer (Scott and Lawrence, 1997), dried fruits ( Abdul Kadar et al., 2004, Arrus et al. (2004), milk and dairy products (Kamkar, 2004; Aycicek et al. 2005; Sarimehmetoglu et al., 2004; Martin and Martin, 2004 ). Meat and meat products are also contaminated with alfatoxins when farm animals are fed with aflatoxin contaminated feed (Miller, 1987 and Chiavaro et al., 2001). The molds that are major producers of aflatoxin are Aspergillus flavus (Bankole et al. 2004) and Aspergillus parasiticus (Begum and Samajpati, 2000; Setamou et al., 1997; Erdogen, 2004; Gourama and Bullerman, 1995). Aspergillus flavus, which is ubiquitous, produces B aflatoxins (Samajphati, 1979) while Aspergillus parasiticus, which produces both B and G aflatoxins, has more limited distribution (Garcia-Villanova et al., 2004). A picture of Aspergillus flavus seen under an electron microscope is shown in Figure 1.0. Black olive is one of the substrate for Aspergillus parasiticus growth and aflatoxin B1 production as reported by Leontopoulos et al., (2003). Biosynthesis of aflatoxins by this fungi depends on the environmental condition such as temperature and humidity during crop growth and storage (Leszczynska et al., 2000; Tarin et al.,

4 2004 and Pildain et al., 2004). The optimum temperatures for aflatoxins growth are 27.84 0 C and 27.30 0 C at pH=5.9 and 5.5 respectively.

Figure 1.0:

Aspergillus flavus seen under an electron microscope

Before harvest, the risk for the development of aflatoxins is greatest during major drought (Turner et al., 2005). When soil moisture is below normal and temperature is high, the number of Aspergillus spores in the air increases. These spores infect crops through areas of damage caused by insects and inclement weather. Once infected, plant stress occurs, which favor the production of aflatoxins. Fungal growth and aflatoxins contamination are the sequence of interactions among the fungus, the host and the environment. The appropriate combination of water stress, high temperature stress and insect damage of the host plant are major determining factors in mold infestation and toxin production (Faraj et al., 1991; Koehler, 1985; Park and Bullerman, 1983). Additional factors such as heat treatment, modified-atmosphere packaging or the presence of preservative, also contribute in increasing growth rate of the aflatoxins. Farmers have minimal control over some of these environmental factors. However appropriate pre-harvest and post-harvest management and good agricultural practice, including crop rotation, irrigation, timed planting and harvesting and the use

5 of pesticides are the best methods for preventing or controlling aflatoxins contamination (Turner et al., 2005). Timely harvesting could reduce crop moisture to a point where the formation of the mould would not occur. For example harvesting corn early when moisture is above 20 percent and then quickly drying it to a moisture level of at least 15 percent will keep the Aspergillus flavus from completing its life cycle, resulting in lower aflatoxin concentration. Aflatoxins are to be found in agricultural products as a consequence of unprosperous storage conditions where humidity of 70 90 % and a minimum temperature of about 10° C. Commodities that have been dried to about 12 to 0.5 % moisture are generally considered stable, and immune to any risk of additional aflatoxins development. Moreover, the minimum damage of shells during mechanized harvesting of crop reduces significantly the mould contamination. Biocontrol of aflatoxin contamination is another way to reduce this contamination. The natural ability of many microorganisms including bacteria, actinomycetes, yeasts, moulds and algae has been a source for bacteriological breakdown of mycotoxins. The most active organism such as Flavobacterium aurantiacum which in aqueous solution can take up and metabolise aflatoxins B1, G1 and M1 (Smith and Moss, 1985). Production of aflatoxins is greatly inhibited by propionic acid as revealed by Molina and Gianuzzi (2002) when they studied the production of aflatoxins in solid medium at different temperature, pH and concentration of propionic acid. It also can be inhibited by essential oil extracted from thyme as found by Rasooli and Abnayeh (2004). Other chemicals that can inhibit the growth of this fungus are ammonia, copper sulphate and acid benzoic (Gowda et al., 2004).

1.2.2

Chemistry of Aflatoxins Aflatoxins can be classified into two broad groups according to chemical

structure which are difurocoumarocyclopentenone series and ifurocoumarolactone (Heathcote, 1984). They are highly substituted coumarin derivatives that contain a fused dihydrofuran moiety. The chemical structure of coumarin is shown in Figure 1.1.

6 O

O

Figure 1.1

Chemical structure of coumarin

There are six major compounds of aflatoxin such as aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG2), aflatoxin G2 (AFG2), aflatoxin M1 (AFM1) and aflatoxin M2 (AFM2) (Goldblatt, 1969). The former four are naturally found aflatoxins and the AFM1 and AFM2 are produced by biological metabolism of AFB1 and AFB2 from contaminated feed used by animals. They are odorless, tasteless and colorless. The scientific name for these aflatoxin compounds are listed in Table 1.0. Aflatoxins have closely similar structures and form a unique group of highly oxygenated, naturally occurring heterocyclic compounds. The chemical structures of these aflatoxins are shown in Figure 1.2. The G series of aflatoxin differs chemically from B series by the presence of a β-lactone ring, instead of a cyclopentanone ring. Also a double bond that may undergo reduction reaction is found in the form of vinyl ether at the terminal furan ring in AFB1 and AFG1 but not in AFB2 and AFG2. However this small difference in structure at the C-2 and C-3 double bond is associated with a very significant change in activity, whereby AFB1 and AFG1 are carcinogenic and considerably more toxic than AFB2 and AFG2. The dihydrofuran moiety in the structure is said to be of primary importance in producing biological effects. Hydroxylation of the bridge carbon of the furan rings for AFM1 does not significantly alter the effects of the compounds. The absolute configuration of AFB2 and AFG2 follows from the fact that it is derived from the reduction of AFB1 and AFG1 respectively. AFB is the aflatoxin which produces a blue color under ultraviolet while AFG produces the green color. AFM produces a blue-violet fluorescence while AFM2 produces a violet fluorescence (Goldblatt, 1969). Relative fluorescence of aflatoxins in several organic solvents are shown in Appendix A (White and Afgauer, 1970). The

7 Table 1.0

Scientific name for aflatoxin compounds

Aflatoxin B1

2,3,6a,9a-tetrahydro-4-methoxycyclopenta[c]

(AFB1)

furo[3’,2’:4,5]furo[2,3-h][l] benzopyran-1,11-dione

Aflatoxin B2

2,3,6a,8,9,9a-Hexahydro-4-methoxycyclopenta[c]

(AFB2)

furo[3’,2’:4,5]furo[2,3-h][l] benzopyran-1,11-dione

Aflatoxin G1 3,4, 7a,10a-tetrahydro-5-methoxy-1H, 12H (AFG1)

furo[3’,2’:4,5]furo[2,3-h]pyrano[3,4-c]l]- benzopyran1,12-dione

Aflatoxin G2 3,4,7a,8,9,10, 10a-Hexahydro-5-methoxy-1H,12H(AFG2)

furo[3’,2’:4,5]furo[2,3-h]pyrano[3,4-c][l]- benzopyran1,12-dione

Aflatoxin

4-Hydroxy AFB1

M1 (AFM1) Aflatoxin

4-Hydroxy AFB2

M2

natural fluorescence of aflatoxins arises from their oxygenated pentaheterocyclic structure. The fluorescence capacity of AFB2 and AFG2 is ten times larger than that of AFB1 and AFG1, probably owing to the structural difference, namely double bond on the furanic ring. Such a double bond seems to be very important for the photophysical

8

O

O

O

O

O

O

O

O

O

O

O

O

(a)

(b)

O

O

O

O

O

O

O

O

O

O

O

O

O

O

(c) O

(d) O O

O

O O

OH

OH

O

O

O

O

O

O

(e) Figure 1.2

(f)

Chemical structures of (a) AFB1, (b) AFB2, (c) AFG1, (d) AFG2,

(e) AFM1 and (f) AFM2. properties of these derivatives measured just after spectroscopic studies (Cepeda et al., 1996). The excitation of the natural fluorescence of AFB1 and AFG1 can be promoted

9

in many different ways such as post-column iodination (Tuinstra and Haasnoot, 1983; Davis and Diener, 1980), post-column bromination (Kok et al.,1986; Kok, 1994 ; Versantroort et al., 2005 ), use of cyclodextrin compound (Cepeda et al., 1996; Chiavaro et al., 2001; Franco et al., 1998) and trifluoroacetic acid, TFA (Stack and Pohland, 1975; Takahashi, 1977a; Haghighi et al., 1981; Nieduetzki et al., 1994 ). AFB1 and AFG1 form hemiacetals, AFB2a and AFG2a when reacted with acidic solution such as triflouroacetic acid (TFA) as represented in Figure 1.3 (Joshua, 1993). The hydroxyaflatoxins are unstable and tend to decompose to yellow products in the presence of air, light and alkali. Their UV and visible spectra are similar to those of the major aflatoxins.

O

O O

O

O O

TFA HO O

O

O

O

O

O

(a) O

(c)

O

O

O O

O

TFA

O

O

HO

O

O O

O

(b) Figure 1.3

O

O

(d)

Hydration of AFB1 (a) and AFG1 (b) by TFA produces AFB2a (c) and

AFG2a (d) (Joshua, 1993). The close relationship between AFB1, AFG1, AFB2a and AFG2a was shown by the similarities in their IR and UV spectra. The main difference between AFB2a and AFG2a with AFB1 and AFG1 are found in the IR spectra, where an additional band at

10 3620 cm-1 indicates the presence of a hydroxyl group in AFB2a and AFG2a. The absence of bands at 3100, 1067 and 722 cm-1 (which arise in AFB1 and AFG1 from the vinyl ether group) indicates that the compounds are hydroxyl derivatives of AFB2 and AFG2. Some chemical and physical properties of aflatoxin compounds are listed in Table 1.1 (Heathcote and Hibbert, 1978; Weast and Astle, 1987). The close relationship between these aflatoxins was shown by the similarities in their UV and IR spectra as shown in Appendix B and C respectively.

Table 1.1

Chemical and physical properties of aflatoxin compounds

AFB1

AFB2

AFG1

AFG2

Molecular formula

C17H12O6

C17H14O6

C17H12O7

C17H14O7

Molecular weight

312

314

328

330

Crystals

Pale yellow

Pale yellow

Colorless

Colorless

Melting point (°C)

268.9

286.9

244.6

237.40

Fluorescence under UV light

Blue

Blue

Green

Green

Solubility

Soluble in water and polar organic solvent. Normal solvents are: methanol, water: acetonitrile (9:1), trifluoroacetic acid, methanol: 0.1N HCl (4:1), DMSO and acetone

Other properties

Odorless, colorless and tasteless in solution form. Incompatible with strong acids, strong oxidising agents and strong bases. Soluble in water, DMSO, 95% acetone or ethanol for 24 hours under ambient temperature

11 Many researches have studied the stability of aflatoxins. For example, AFB1 was not found in fruit samples after being irradiated with 5.0 Kgy or more of gammairradiation as reported by Aziz and Moussa (2002). Gonzalez et al. (1998) have studied the effect of electrolysis, ultra-violet irradiation and temperature on the decomposition of AFB1 and AFG1. UV irradiation caused an intense effect on aflatoxins where after 60 min of radiation, AFG1 suffers practically with no more decomposition. However, when the aflatoxin solutions were placed in a 90° C bath for 3 min, a decrease of 20% from total amount of both AFB1 and AFG1 was obtained. A greater extent of decomposition (50%) was found for treatment at 100° C during longer time. Levi (1980) reported that experimental roasting under conditions simulating those of the typical roasting operation (20 min at 200 ± 5° C) destroyed about 80% of AFB1 added to green coffee. It was also found that AFB2 decomposed to a larger extent than AFG1 indicating a lower stability against prolonged heat treatment. During food fermentation which involved other fungi such as Rhizopus oryzae and R. oligosporus, cyclopentanone moiety of AFB1 was reduced resulting in the formation of aflatoxicol A as shown in Figure 1.4 which is non-toxic compound. It retains the blue-fluorescing property of AFB1 under UV light. It has been considered to be one of the most important B1 metabolites because there is a correlation between the presence of this metabolite in animal tissues and body fluids with toxicity of AFB1 in different animals (Lau and Chu, 1983). Aflatoxin solution prepared in water, dimethyl sulphonic acid (DMSO), 95% acetone or ethanol is stable for 24 hours under ambient conditions. AFM1 is relatively stable during pesteuring, sterilisation, preparation and storage of various dairy products (Gurbay et al., 2004).

O

O

O O

OH

O

+ R.oryzae + R. oligosporus OCH 3

O O

Figure 1.4

(a)

OCH3

O O

(b)

Transformation of toxic (a) AFB1 to non-toxic (b) aflatoxicol A

12 1.2.3

Health Aspect of Aflatoxins

Aflatoxins have received greater attention than any other mycotoxins. There are potent toxin and were considered as human carcinogen by The International Agency for Research on Cancer (IARC) as reported in World Health Organisation (WHO)’s monograph (1987). These mycotoxins are known to cause diseases in man and animals called aflatoxicosis (Eaton and Groopman, 1994). Human exposure to aflatoxins is principally through ingestion of contaminated foods (Versantroort et al., 2005). Inhalation of the toxins may also occur occasionally due to the occupational exposure. After intake of contaminated feedstuffs aflatoxins cause some undesirable effect in animals, which can range from vomiting, weight loss and acute necrosis to various types of carcinoma, leading in many cases to death (Pestka and Bondy, 1990 and Bingham et al., 2004). Even at low concentration, aflatoxins diminish the immune function of animals against infection. Epidemiological studies have shown a correlation between liver cancer and the prevalence of aflatoxins in the food supply. In views of occurrence and toxicity, AFB1 is extremely carcinogenic while others are considered as highly carcinogenic as reported by Carlson (2000). It is immunosuppressive and a potent liver toxin; less than 20 µg of this compound is lethal to duckling (Hussein and Brasel, 2001). AFB1 is biochemically binding to DNA, inhibit DNA, RNA, and protein syntheses, and effects DNA polymerase activity as reported by Egner et al. (2003). Previous research results have demonstrated the covalent binding of highly reactive metabolite of AFB1 to the N-7 atom of guanine residues, resulting in major DNA adducts (Moule, 1984; Johnson et al., 1997; Egner et al., 2003). Major DNA adducts of AFB1 are shown in Figure 1.5.

The four main aflatoxins display decreasing potency in the order AFB1 > AFG1 > AFB2 > AFG2 as reported by Betina (1984). This order of toxicity indicates that the double bond in terminal furan of AFB1 structure is a critical point for determining the degree of biological activity of this group of mycotoxins (Hall and Wild, 1994). It appears that the aflatoxins themselves are not carcinogenic but rather some of their

13

O

O

O

O

O OH

OH O

O

N

NH

NH2

O

O

N

N

O

N

OCH3

NH

NH2

(a) Figure 1.5

O

O

OCH3

CHO

N

NH2

(b)

Major DNA adducts of AFB1; (a) 8,9- Dihydro-8-(N7-guanyl)-

9-hydroxy-aflatoxin B1 (AFB1-Gua) and (b) 8,9-Dihydro-8-(N5- Formyl-2’.5’.6 ’triamino-4 ’-oxo-N5-pyrimidyl)-9-hydroxy- Aflatoxin B1 (AFB1-triamino-Py)

metabolites (de Vries, 1996). For example, metabolite transformation of AFB1 by cytochrome P-450 enzyme produces aflatoxin Q1 (AFQ1), AFM1, AFB1-epoxide and aflatoxin P1 (AFP1) as shown in Figure 1.6. For hamiacetals, AFB2a and AFG2a, however, are relatively non-toxic despite the close similarity of their structure to those of AFB1 and AFG1 even at the highest dosages (1200 µg for AFB2a and 1600 µg for AFG2a). AFM1 is the main metabolic derivatives of aflatoxins in several animal species It is found in the cow milk due which had consumed feed which contaminated with AFB1 as reported by Chang et al. (1983), Yousef and Marth (1985), Van Egmond (1989), Tuinstra (1990) and Lopez et al. (2002). The relative amount of AFM1 excreted is related to the amount of AFB1 in the feed, and about 0.1% of AFB1 ingested is excreted into milk as AFM1 (Miller, 1987). There was a linear relationship between the amount of AFM1 in milk and AFB1 in feeds consumed by animals as reported by Dragacci (1995). AFM1 is produced by hydroxylation of AFB1 in the liver

14

O

O

O

O O

O

OCH3

B 1 -e p o x id e

O

O

O

O

O

OCH3

A flato x in B 1 O

O

O

O

O

O

O

O

O

OH

OH

O

O

O

M1 OCH3

Figure 1.6

O

O

P1

O

QI OH

Metabolic pathways of AFB1 by cytochrom P-450 enzymes; B1-

epoxide = AFB1 epoxide, M1 = aflatoxin M1, P1 = aflatoxin P1 and Q1 = aflatoxin Q1

OCH3

15 of lactating animals, including humans. It is also known as milk toxin which is much less carcinogenic and mutagenic than AFB1. It has been classified by the International Agency For Research Cancer (IARC) as a Group 2 carcinogen (IARC, 1993). It can generally be found in milk and milk products such as dry milk, whey, butter, cheese, yogurt and ice cream. AFM2 is the analogous metabolic derivatives of AFB2. Aflatoxins have been considered as one of the most dangerous contaminant in food and feed. The contaminated food will pose a potential health risk to human such as aflatoxicosis and cancer (Jeffrey and Williams, 2005). Aflatoxins consumption by livestock and poultry results in a disease called aflatoxicosis which clinical sign for animals include gastro intestinal dysfunction, reduced reproductivity, reduced feed utilisation, anemia and jaundice. Humans are exposed to aflatoxins by ingestion, inhalation and dermal exposure as reported by Etzel (2002). LD50 which is the amount of a materials, given all at once, causes the death of 50% (one half) of a group of test animal (www.aacohs.ca/oshanswers) for most animal and human for AFB1 is between 0.5 to 10.0 mg kg-1 body weight (Smith and Moss, 1985; Salleh, 1998). Clinical features were characterised by jaundice, vomiting, and anorexia and followed by ascites, which appeared rapidly within a period of 2-3 weeks. Besides causing health problems to humans, aflatoxin also can cause adverse economic effect in which it lowers yield of food production and fiber crops and becoming a major constraint of profitability for food crop producer countries, an example of which is given in Rachaputi et al. (2001). It has been estimated that mycotoxin contamination may affect as much as 25% of the world’s food crop each year (Lopez, 1999) resulting in significant economic loss for these countries. Aflatoxins also inflict losses to livestock and poultry producers from aflatoxincontaminated feeds including death and the more subtle effects of immune system suppression, reduced growth rates, and losses in feed efficiency. Due to the above reason, aflatoxin levels in animal feed and various human food products is now monitored and tightly regulated by the most countries. In Malaysia,

16 the action level for total aflatoxins is 15ppb (Malaysian Food Act, 1983). The European Commission has set limits for the maximum levels of total aflatoxins and AFB1 allowed in groundnuts, nuts, dried fruit and their products. For foods ready for retail sale, these limits are 4 ppb for total aflatoxins and 2 ppb for AFB1, and for foods that are to be processed further the limits stand at 15 ppb for total aflatoxins and 8 ppb of AFB1 (European Commission Regulation, 2001). The Food and Drug Administration (FDA) in USA has established an action level of 0.5 ppb of mycotoxin, AFM1 in milk for humans and 20 ppb for other aflatoxins in food other than milk (www.ansci.cornel.edu/toxiagents). In Brazil, the limits allowed for food destined for human consumption are 20 µg/kg of total aflatoxin for corn in grain, flours, peanut and by-products, and 0.5 µg/l of AFM1 in fluid milk (Sassahara et al., 2005). Australia has established a minimum level of 15 ppb for aflatoxin in raw peanut and peanut product (Mackson et al., 1999). In Germany, regulatory levels of total aflatoxins are 4 ppb and 2 ppb for AFB1. For human dietary products, such as infant nutriment, there are stronger legal limits, 0.05 ppb for AFB1 and the total aflatoxins (Reif and Metzger, 1995). The Dutch Food Act regulates that food and beverages may contain no more than 5 µg of AFB1 per kg (Scholten and Spanjer, 1996). Regulatory level set by Hong Kong government is 20 ppb for peanuts and peanut products and 15 ppb for all other foods (Risk Assessment Studies report, 2001). Because of all these reasons, systematic approaches to sampling, sample preparation and selecting appropriate and accurate method of analysis of aflatoxin are absolutely necessary to determine aflatoxins at the part-of-billion (ppb) level as reported by Park and Rua (1991). 1.2.4

Analytical Methods for the Determination of Aflatoxins

Monitoring the presence of aflatoxin in various samples especially in food is not only important for consumer protection, but also for producers of raw products

17 prior to cost intensive processing or transport. Several methods for aflatoxins determination in various samples have been developed and reported in the literature. Method based on thin-layer chromatography (TLC) (Lafont and Siriwardana, 1981; AOAC, 1984) and high performance liquid chromatography (HPLC) with ultraviolet absorption, fluorescence, mass spectrometry or amperometry detection, have been reported (Kok et al., 1986; Taguchi et al., 1995; Ali et al., 2005; Manetta et al., 2005). TLC and HPLC techniques are well proven and widely accepted; however, both techniques have their own disadvantages. These methods require well equipped laboratories, trained personnel, harmful solvents and time consuming. Moreover, instrumentations used are expensive (Badea et al., 2004). The main disadvantage associated with TLC is the lack of precision where a possible measurement error of ± 30 – 50 % is indicated when standard and unknown aflatoxins spot are matched, and ± 15 -25 % when the unknown is interpolated between two standards (Takahashi, 1977b). In this technique, the greater effect was caused by sample matrix which is usually very complicated (Lin et al., 1998). Poor repeatability is associated with the sample application, development and plate interpretation steps. HPLC technique is often viewed as laborious and time intensive, needs complex gradient mobile phase, large quantity of organic solvent, requiring a significant investment in equipments, materials and maintenance (Pena et al., 2002). Pre-column derivatisation technique could improve the sensitivity of the measurement however, it requires chemical manipulations which are time consuming, involves aggressive reagents such as bromine, TFA and iodine and also it is difficult to automate. Other disadvantages of this procedure include the requirement to prepare iodine solution daily, the necessity for two pumps, dilution of the eluent stream, the need to thermostat the reactor coil and insufficient day-to-day reproducibility (Kok et al., 1986). The method using TFA as derivatising agent has poor reproducibility and is difficult to automate (Reif and Metzger, 1995).

18 Other methods such as enzyme linked immunosorbant assay (ELISA) (Chu et al., 1987; Lee et al., 1990; Pesavento et al., 1997 and Aycicek et al., 2005),

radioimmunoassay (RIA) (Stroka et al., 2000) and immunoaffinity clean up (Garner, 1993 and Niedwetzki et al., 1994) have also been developed for the detection of these compounds. The simplicity, sensitivity and rapid detection of aflatoxin by ELISA has made it possible to monitor several samples simultaneously but ELISA and other immunochemical methods require highly specific polyclonal or monoclonal sera for specific and sensitive detection of antigen. The production of specific antibodies for aflatoxins has allowed the development of this technique based on direct or indirect competition. This method, however, presents some drawbacks such as long incubation time, washing and mixing steps (Badea et al., 2004) and is labor intensive (Carlson et al., 2000). It also requires highly specific polyclonal or monoclonal sera which is expensive (Rastogi et al., 2001). Blesa et al. (2003) reported that the ELISA method is a good screening method for investigation of aflatoxins. RIA is very sensitive but has several disadvantages. The radioisotopes are health hazards, disposable difficulties and may have short half life (Trucksess et al., 1991). Cole et al., (1992) have used micellar electrokinetic capillary chromatography (MECC) technique for rapid separation of aflatoxins. Pena et al., (2002) proposed capillary electrophoresis (CE) for determination of aflatoxins. They found that the limit of detection (LOD) of this technique was 0.02 to 0.06 ppb with analysis time of 50 minutes. Due to long duration time and involving use of many reagents such as benzene and acetonitrile for preparation of aflatoxin stock solution and sodium dodecyl sulphate (SDS), sodium dihydrogenphosphate, sodium borate and γcyclodextrin for preparing the buffer solution in performing the analysis, this technique may not be suitable for routine analysis of aflatoxins. A summary of techniques used for the determination of aflatoxins compounds in various samples together with its detection limit is shown in Table 1.2.

19 Table 1.2

Summary of analysis methods used for the determination of aflatoxins in

various samples No

Method

Aflatoxin / samples

Detection limit (ppb)

Reference

1

HPLC-FD

B1, B2, G1 and G2 in spices

0.06

Garner et al. 1993)

2

HPLC-FD

B1, B2, G1 and G2 in spices

0.5

Akiyama et al. (2001)

3

HPLC-FD

B1, B2, G1 and G2 in peanut butter

5.0

Beebe (1978)

4

HPLC-FD

B1, B2, G1 and G2 in peanut butter

0.5

Duhart et al. (1988)

5

HPLC-FD

B1, B2, G1 and G2 in peanut butter

Not stated

Patey et al. (1991)

6

HPLC-FD

B1, B2, G1 and G2 in airborne dust

B1, G1, G2 Kussak et al. = 3.1 (1995a) B2 = 1.8

7

HPLC-FD

B1, B2, G1 and G2 in corn, peanuts and peanut butter

Not stated

Trucksess et al. (1991)

8

HPLC-FD

B1, B2, G1 and G2 in medicinal herbs and plant extract

0.05

Reif and Metzger (1995)

9

HPLC-FD

B1, B2, G1 and G2 in airborne dust and human sera

0.08

Brera et al. (2002)

20 Table 1.2

No

Method

Continued

Aflatoxin / samples

Detection limit (ppb)

Reference

2.0

Chemistry Department, MOSTI (1993)

Not stated

Holcomb et al. (2001)

10

HPLC-FD

B1, B2, G1 and G2 in groundnut, peanut and peanut butter

11

HPLC-FD

B1, B2, G1 and G2 in standard samples

12

HPLC-FD

B1, B2, G1 and G2 in wine

0.02

13

HPLC-FD

B1, B2, G1 and G2 in corn, almonds, peanuts, milo,rice, pistachio, walnuts, corn and cottonseed

Not stated

14

HPLC-FD

M1 in milk and non fat dry milk

0.014

Chang and Lawrence (1997)

15

HPLC-FD

B1, B2, G1 and G2 in beer

B1, G1= 0.019, B2, G2 = 0.015

Scot and Lawrence (1997)

16

HPLC-FD

B1, B2, G1 and G2 in poultry and pig feeds and feedstuffs

1.0

Cespedes and Diaz (1997)

17

HPLC-FD

M1 in milk

0.05

Yousef and Marth (1985)

18

HPLC-FD

M1 in whole milk

0.014

Fremy and Chang (2002)

Takahashi (1977a) Wilson and Romer (1991)

21 Table 1.2

No

Method

Continued

Aflatoxin / samples

Detection limit (ppb)

Reference

19

HPLC-FD

B1, B2, G1 and G2 in pistachio B1 = 0.8, kernels and shells B2 = 0.29 G1 = 0.9 G2 = 1.1

Chemistry Department, MOSTI (1993)

20

HPLC-FD

B1, B2, G1 and G2 in urine

0.0068

Kussak et al. (1995b)

21

HPLC-FD

M1 in milk

10

Gurbey et al. (2004)

22

HPLC-FD

M1 in urine and milk

23

HPLC-FD

B1, B2, G1 and G2 in sesame butter and tahini

24

HPLC-FD

25

0.0025

Simon et al. (1998)

Not stated

Nilufer and Boyacio (2002)

B1 and ochratoxin A in bee pollen

B1= 0.2

GarciaVillanove et al. (2004)

HPLC-FD and UVD

B1, B2, G1 and G2 in contaminated cocoa beans

1.0

Jefferey et al. (1982)

26

HPLC-FD and UVD

B1 and B2 in wine and fruit juices

0.02

Takahashi (1997b)

27

HPLCUVD

B1, B2, G1 and G2 in corn

B1, G1 = 1.0, B2, G2 = 0.25

Fremy and Chang (2002)

22 Table 1.2

Continued

Aflatoxin / samples

Detection limit (ppb)

Reference

Not stated

Rastogi et al. (2001)

No

Method

28

HPLCUVD

B1in standard sample

29

HPLCUVD

B1and B2 in cottonseed

5.0

Pons and Franz (1977)

30

HPLCUVD

B1 in egg

1.0

Trucksess et al. (1977)

31

HPLCUVD

B1 and M1 in corn

B1 = 3.0 -5.0 M1 = not stated

Stubblefield and Shotwell (1977)

32

HPLCUVD

B1, B2, G1 and G2 in soy sauces and soybean paste

33

HPLC-AD B1, B2, G1 and G2 in standard sample

34

HPTLC

35

36

1.0

Wet et al. (1980)

7.0

Gonzalez et al. (1998)

B1, B2, G1 and G2 in corn, buckwheat, peanuts and cheese

B1 = 0.2 B2, G1, G2 = 0.1

Kamimura et al. (1985)

HPTLCSPE

B1, B2, G1 and G2 in palm kernel

B1 = 3.7, B2 = 2.5, G1 = 3.0, G2 = 1.3

Nawaz et al. (1992)

TLC

B1and M1in artificial contaminated beef livers

B1= 0.03, M1 = 0.1

Stabblefield et al. (1982)

23 Table 1.2

Continued

Aflatoxin / samples

Detection limit (ppb)

Reference

TLC

B1in milk and milk powder

0.05

Bijl and Peteghem (1985)

38

TLC

Total aflatoxin in wheat and soybean

Not stated

Shotwell et al. (1977b)

39

TLC

B1, B2, G1 and G2 in apples, pears, apple juice and pear jams

B1, G1 = 2.02.8, B2, G2 = 2.0

Gimeno and Martins (1983)

40

TLC

B1, B2, G1 and G2 in corn and peanut meal

Corn = 2.0 Bicking et al. Peanut meal = (1983) 3.0

41

TLC

M1 in milk and condensed, evaporated and non-fat powdered milk

42

TLC

B1, B2, G1 and G2 in ginger root and oleoresin

Not stated

Trucksess and Stoloff (1980)

43

TLC

B1in black olives

3000-7000

Tutour et al. (1984)

44

TLC

B1, B2, G1 and G2 in cereal and nuts

Not stated

Saleemullah et al. (1980)

45

TLC-FDs

No

Method

37

M1in milk

Milk = 0.1 Others = 0.2

0.005

Fukayama et al. (1980)

Gauch et al. (1982)

24 Table 1.2

No

46

Method

Continued

Aflatoxin / samples

OPLC-FD B1, B2, G1 and G2 in maize, fish, wheat, peanuts, rice and sunflower seeds

Detection limit (ppb)

Reference

Not stated

Papp et al. (2002)

1.0

Vahl and Jargensen (1998)

47

LC-MS

B1, B2, G1 anf G2 in figs and peanuts

48

ELISA

M1 in milk, yogurt, cheddar and Brie

0.01 – 0.05

Fremy and Chu. (1984)

49

ELISA

M1 in infant milk products and liquid milk

Not stated

Rastogi et al. (1977)

50

ELISA

M1in curd and cheese

Not stated

Grigoriadou et al. (2005)

51

ELISA

M1 in cheese

Not stated

Sarimehtoglu et al. (1980)

52

ELISA

B1, B2, G1 and G2 in sesame butter and tahini

Not stated

Nilufar and Boyacio (2000)

53

ELISA ST B1in corn

54

MECC

B1, B2, G1 and G2 in standard samples

5.0 0.05 – 0.09

Beaver et al. (1991) Cole et al. (1992)

25 Table 1.2

No

55

Method

Continued

Aflatoxin / samples

MECC FS B1, B2, G1 and G2 in feed samples

56

IAC

Total aflatoxins and B1 in nuts

57

IAC

B1 in mixed feeds

58

FI-IA-AD

59

GC-FID

60

ECS with BLMs

61

M1 in raw milk

Detection limit (ppb)

Reference

0.02 – 0.06

Pena et al. (2002)

Total = 1.0 B1 = 0.2

Leszezynska et al. (1998)

2.0

Shannon et al. (1984)

0.011

Badea et al. (1977)

B1, B2, G1 and G2 in culture medium

Not stated

Goto et al. (2005)

M1 in skimmed milk

761

Androu et al. (1997)

DPP

B1 in rice, milk, corn and peeletised rabbit feed

25

Smyth et al. (1979)

62

SIIA

M1in an artificial contaminated food

0.2

Garden and Strachen (2001)

63

MS

B1, B2, G1 and G2 in contaminated corns

10

Plattner et al. (1984)

26 Table 1.2

Continued

Method

64

IAFB

65

IACLC

B1 and total aflatoxin in peanut butter, pistachio paste, fig paste and paprika powder

66

IACLC

B1, B2, G1 and G2 in maize and peanut butter

67

ICA

B1 in rice, corn and wheat

68

TLC

B1, B2, G1 and G2 in peppers

Not stated

Erdogan (2004)

HPLC-FD B1, B2, G1 and G2 in traditional herbal medicines

Not stated

Ali et al. (2005)

0.02 – 0.15

Sorensen and Elbaek (2005)

3.75

Edinboro and Karnes (2005)

0.0125

Bakirci (2001)

69

Aflatoxin / samples

B1in standard sample

70

LC-MS

M1 in bovine milk

71

LC-MS

M1in sidestream cigarette smoke

72

TLC

M1in milk and milk products

Detection limit (ppb)

Reference

No

0.1

Carlson et al. (2002)

Not stated

Stroka et al. (2000)

B1, B2, G1, G2 = 2.0

Chan et al. (1984)

2.5

Xiulan et al. (2006)

27 Table 1.2

Continued

No

Method

Aflatoxin / samples

Detection limit (ppb)

Reference

73

ELISA

B1and M1 in food and dairy products

Not stated

Aycicek et al. (2005)

74

TLC

M1 in Iranian feta cheese

0.015

Kamkar et al. (2005)

75

Ridascreen Test

M1 in pasteurised milk

Not stated

Alborzi et al. (2005)

76

Ridascreen Test

M1 in raw milk

77

TLC

78

B1in melon seeds

ECS-ELISA B1in barley

0.245

Sassahara et al. (2005)

2.0

Bankole et al. (2005b)

0.02 – 0.03

Ammida et al. (2004)

Not stated

Gonzalez et al. (2005)

79

HPLC-FD

B1, B2, G1 and G2 in bee pollen

80

HPLC-FD

M1in milk and cheese

Milk = 0.001, Cheese = 0.005

Manetta et al. (2005)

81

LC-MS

B1, B2, G1 and G2 in peanuts

B1, B2, G1, G2 = 0.125 – 2.50

Blesa et al. (2001)

82

ELISA-SPE

M1 in milk

0.025

Micheli et al. (2005)

28 Table 1.2

Continued

Notes:

AD: IAC: IACLC: IAFB: BLMs: DPP: ECS: ECS-ELISA: ELISA: ELISA ST: ELISA-SPE: FD: FI-IA-AD: GC-FID: HPLC: HPTLC: HPTLC-SPE: ICA: MECC: MS: OPLC: SIIA: TLC: TLC-FDs: UVD:

1.2.5

Amperometric detector Immunoaffinity chromatography Immunoaffinity column liquid chromatogtaphy Immunoaffinity fluorometer biosensor Bilayer lipid membranes Differential pulse polarography Electrochemical sensing Electrochemical sensing based on indirect ELISA Enzyme linked immunosorbant assay Enzyme linked immunosorbant assay for screening Enzyme linked immuno sorbant assay combined with screen printed electrode Fluorescence detector Flow-injection immunoassay with amperometric detector Gas chromatography with flame ionization detecter High pressure liquid chromatography High pressure thin liquid chromatography High pressure thin liquid chromatography with solid phase extraction Immnuochromatographic assay Micellar electrokinetic’s capillary chromatography Mass spectrometry Over pressured liquid chromatography Sequential injection immunoassay test Thin layer chromatography Thin layer chromatography with fluorescence densitometer Ultraviolet detector

Electrochemical Properties of Aflatoxins

Polarographic determination of aflatoxin has been studied by Smyth et al. (1979) using DPP technique to determine AFB1, AFB2, AFG1 and AFG2 in food samples using Britton-Robinson buffer solution as the supporting electrolyte. They found that all aflatoxins exhibited similar polarographic behaviour over a pH range 4 –

29 11. They also obtained that the best-defined waves for analytical purposes were in Britton-Robinson buffer at pH 9. Slight differences in the potential of reduction of aflatoxins have been observed owing to their slight differences in structure. They found that the half wave potentials of reduction of the various aflatoxins were AFB1 = -1.26 V, AFB2 = -1.27 V, AFG1 = -1.21 V and AFG2 = -1.23 V (all versus SCE). The limit of detection for AFB1 in pure solutions was about 2 x 10 -8 M (25 ppb). Gonzalez et al. (1998) have studied the cyclic voltammetry of AFG1 in methanol: water (1:1, v/v) solution on different electrodes such as glassy carbon, platinum and gold electrodes. They found that AFG1 gave high peak current at a potential of about +1.2V using glassy carbon electrode compared to other types of working electrode. The electro activity of the studied aflatoxins increased in the order AFG2 < AFB1 < AFB2 < AFG1. This order reflects the ability of the aflatoxins molecules to undergo electrochemical oxidation on glassy carbon electrode. Duhart et al. (1988) have determined all types of aflatoxin using HPLC technique with electrochemical detection. They have used differential-pulse mode at the dropping mercury electrode (DME) with 1 s drop time for detection system, large drop size, current scale of 0.5 µA and modulation amplitude of 100 mV. They have found that using mobile phase which consisted of 62.7% BRB at pH 7, 17.9% methanol and 19.4% acetonitrile, the peak potentials for all aflatoxins were slightly different which were AFB1 = -1.37 V, AFB2 = -1.36 V, AFG1 = -1.30 V and at -1.30 V for AFG2. Due to the fairly close together of peak potentials, a potential of -1.28 V has been selected for detection purposes of these aflatoxins together with HPLC technique. They found that using this technique average percentage recoveries for peanut butter samples (n = 4) which have been spiked with a mixture of the four aflatoxins were AFB1 (76%), AFB2 (77%), AFG1 (87%) and AFG2 (81%). This results show that a major advantage of this technique was it did not require a derivatisation step as is common for fluorescent detection.

30 1.3

Voltammetric Techniques

1.3.1

Voltammetric Techniques in General

Voltammetry is an electrochemical method in which current is measured as a function of the applied potential. It is a branch of electrochemistry in which the electrode potential, or the faradaic current or both are changed with time. Normally, there is an interrelationship between all three of these variables (Bond et al., 1989). The principle of this technique is a measurement of the diffusion controlled current flowing in an electrolysis cell in which one electrode is polarisable (Fifield and Kealey, 2000). In this technique a time dependent potential is applied to an electrochemical cell, and the current flowing through the cell is measured as a function of that potential. A plot of current which is directly proportional to the concentration of an electroactive species as a function of applied potential is called a voltammogram. The voltammogram provides quantitative and qualitative information about the species involved in the oxidation or reduction reaction or both at the working electrode. Polarography is the earliest voltammetric technique which was developed by Jaroslav Heyrovsky (1890-1967) in the early 1920s, for which he was awarded the Nobel Prize in Chemistry in 1959. It was the first major electro analytical technique (Barek et al., 2001a). Since then many different forms of voltammetry have been developed such as direct current polarography (DCP), normal polarography (NP), differential pulse polarography (DPP), square-wave polarography (SWP), alternate current polarography (ACP), cyclic voltammetry (CV), stripping voltammetry (SV), adsorptive stripping voltammetry (AdSV) and adsorptive catalytic stripping voltammetry (AdCSV) techniques. Working electrodes and limit of detection (LOD) for these modern voltammetric techniques are shown in Table 1.3 (Barek et al., 2001a). The advantage of this technique include high sensitivity where quantitative and qualitative determination of metals, inorganic and organic compounds at trace levels, 10-4 – 10-8 M (Fifield and Kealey, 2000), selectivity towards electro active species

31 (Barek et al., 2001b), a wide linear range, portable and low-cost instrumentation, speciation capability and a wide range of electrode that allow assays of many types of samples such as environmental samples (Zhang et al., 2002; Ghoneim et al., 2000; Buffle et al., 2005), pharmaceutical samples (Yaacob, 1993; Yardimer et al., 2001; Abdine et al., 2002; Hilali et al., 2003 and Carapuca et al., 2005 ), food samples (Ximenes et al., 2000; Volkoiv et al., 2001); Karadjova et al., 2000; Sabry and Wahbi, 1999 and Sanna et al., 2000), dye samples (Mohd Yusoff et al., 1998 and Gooding et al., 1997) and forensic samples (Liu et al., 1980; Wasiak et al., 1996; Pourhaghi-Azar

and Dastangoo, 2000; Woolever et al., 2001).

Various advances during the past few years have pushed the detectability of voltammetric techniques from the submicromolar level for pulse voltammetric techniques to the subpicomolar level by using an adsorptive catalytic stripping voltammetry (Czae and Wang, 1999). The comparison of using polarographic and other analytical techniques in different application fields is depicted in Table 1.4 (Barek et al., 2001a).

1.3.2

Voltammetric Measurement

1.3.2.1 Instrumentation

Voltammetry technique makes use of a three-electrode system such as working electrode (WE), reference electrode (RE) and auxiliary electrode (AE). The whole system consist of a voltammetric cell with a various volume capacity, magnetic stirrer and gas line for purging and blanketing the electrolyte solution.

32 Table 1.3

Working electrodes and LOD for modern polarographic and

voltametric techniques

Technique

Working

LOD

electrode

TAST

DME

~ 10 -6 M

Normal pulse polarography (NPP)

DME

~ 10 -7 M

Normal pulse voltammetry (NPV)

HMDE

~ 10 -7 M

Stair case voltammetry (SCV)

HMDE

~ 10 -7 M

Differential pulse polarography (DPP)

DME

~ 10 -7 M

Differential pulse voltammetry (DPV)

HMDE

~ 10 -8 M

Square wave polarography (SWP)

DME

~ 10 -8 M

Square wave voltammetry (SWV)

HMDE

~ 10 -8 M

Alternate current polarography (ACP)

DME

~ 10 -7 M

Alternate current voltammetry (ACV)

HMDE

~ 10 -8 M

Anodic stripping voltammetry (ASV)

HMDE

~ 10 -10 M

Cathodic stripping voltammetry (CSV)

MFE

~ 10 -9 M

Potentiometric stripping analysis (PSA)

MFE

~ 10 -12 M

33 Table 1.4

The application range of various analytical techniques and their

concentration limits when compared with the requirements in different fields of chemical analysis.

Field of chemical analysis

Concentration range

Environmental monitoring

10-12 M to 10 -4 M

Toxicology

10-11 M to 10 -2 M

Pharmacological studies

10-10 M to 10 -4 M

Food control

10-8 M to 10-4 M

Forensic

10-7 M to 10-3 M

Drug assay

10-5 M to 10 -2 M

Analytical techniques

Application range

Adsorptive stripping voltammetry

10-12 M to 10-7 M

Anodic / cathodic stripping voltammetry

10-11 M to 10-6 M

Differential pulse voltammetry

10-8 M to 10-3 M

Differential pulse polarography

10-7 M to 10-3 M

Tast polarography

10-6 M to 10-3 M

d.c.polarography

10-5 M to 10-3 M

spectrophotometry

10-6 M to 10-3 M

HPLC with voltammetric detection

10-7 M to 10-3 M

HPLC with fluorescence detection

10-9 M to 10-3 M

34 Table 1.4

Continued

Analytical techniques

Application range

Spectrofluorometry

10-9 M to 10-3 M

Atomic absorption spectrometry

10-8 M to 10-3 M

Atomic fluorescence spectrometry

10-9 M to 10-3 M

Radioimmunoanalysis

10-13 M to 10 -3 M

Neutron activation analysis

10-9 M to 10-3 M

x-ray fluorescence analysis

10-7 M to 10-3 M

mass spectrometry

10-9 M to 10-3 M

A typical arrangement for a voltammetric electrochemical cell is shown in Figure 1.7 (Fifield and Haines, 2000). N2 Inlet RE

WE

AE

Cell cover

Solution Level

Stirrer

Figure 1.7

A typical arrangement for a voltammetric electrochemical cell

(RE: reference electrode, WE: working electrode, AE: auxiliary electrode)

35 The arrangement of the electrodes within the cell is important. The RE is placed close to the WE and the WE is located between the RE and the AE. Using the three-electrode-cell concept, a potentiostat monitors the voltage over the WE and AE which is automatically adjusted to give the correct applied potential. This is obtained by continuously measuring the potential between the WE and the RE, by comparing it to the set voltage and by adjusting the applied voltage accordingly if necessary. The cell material depends on application that it is usually a small glass beaker with a close fitting lid, which includes ports for electrodes and a nitrogen gas purge line for removing dissolved oxygen and an optional stir bar. The WE is the electrode where the redox reaction of electroactive species takes place and where the charge transfer occurs. It is potentiostatically controlled and can minimise errors from cell resistance. It is made of several different materials including mercury, platinum, gold, silver, carbon, chemically modified and screen printed electrode. The performance of voltammetry is strongly influenced by the WE. The ideal characteristics of this electrode are a wide potential range, low resistance, reproducible surface and be able to provide a high signal-to-noise response. The WE must be made of a material that will not react with the solvent or any component of the solution over as wide a potential range as possible. The potential window of such electrodes depends on the electrode material and the composition of the electrolyte as summarised in Table 1.5 below (Wang, 2000). Majority of electrochemical methods use HMDE and MFE (Economou and Fielden, 1995) for use in the cathodic potential area, whereas solid electrode such as gold, platinum, glassy carbon, carbon paste are used for examining anodic processes. Mercury has been used for the WE in earlier voltammetry techniques, including polarography. Since mercury is a liquid, the WE often consists of a drop suspended from the end of a capillary tube. It has several advantages including its high over potential for the reduction of hydronium ion to hydrogen gas. This allows for the application of potential as negative as -1.0 V versus SCE in acidic solution, and -2.0V versus SCE in basic solution.

36 Table 1.5

List of different type of working electrodes and its potential windows.

ELECTRODE

ELECTROLYTE

POTENTIAL WINDOWS

1 M H2SO4 1 M KCl 1 M NaOH 0.1 M Et4NH4 OH

+0.3 0 -0.1 -0.1

Pt

1 M H2SO4 1 M NaOH

+1.0 +0.5

C

1 M HClO4 0.1M KCl

+1.5 to +1.0 to

Hg

to to to to

-0.1V -1.9V -2.0V -2.3V

to -0.5V to -1.0V -1.0V -1.3V

Other advantages of using mercury as the working electrode include the ability of metals to dissolve in the mercury, resulting in the formation of an amalgam. The greatest advantages of this electrode is that new drops or new thin mercury films can be readily formed, and this cleaning process removes problems that could be caused by contamination as a result of previous analysis. In contrast, this is not the generally case for electrodes made from other materials, with the possible exception of carbon electrodes, where the electrode cleaning is made of cutting off a thin layer of the previous electrode surface. Another advantage is the possibility to achieve a state of pseudostationary for linear sweep voltammetry (LSV) using higher scan rate. Miniaturised and compressible mercury electrode offer new possibilities in voltammetry especially for determination of biologically active species and surfactant. One limitation of mercury electrode is that it is easily oxidised at + 0.3 V and cannot be used at potential more positive than + 0.4 V versus the SCE, depending on the composition of the solution (Dahmen, 1986 and Fifield and Haines, 2000). At this potential, mercury dissolves to give an anodic polarographic wave due to formation of mercury (I) (Skoog et al., 1996).

37 There are three main types of mercury electrode used in voltammetry techniques including hanging mercury drop electrode (HMDE), dropping mercury drop electrode (DME) and static mercury drop electrode (SMDE). In the HMDE, a drop of the desired size is formed and hanged at the end of a narrow capillary tube. In the DME, mercury drops form at the end of the capillary tube as a result of gravity. The drop continuously grows and has a finite lifetime of several seconds. At the end of its lifetime the mercury drop is dislodged, either manually or by the gravity, and replaced by a new drop. In the SMDE, it uses a solenoid-driven plunger to control the flow of mercury. It can be used as either HMDE or DME. A single activation of solenoid momentarily lifts the plunger, allowing enough mercury to flow through the capillary to form a single drop. To obtain a dropping mercury electrode the solenoid is activated repeatedly. The diagram of HMDE is shown in Figure 1.8 (Metrohm, 2005).

Figure 1.8

A diagram of the HMDE

38 Other type of mercury electrode is the controlled growth mercury drop electrode (CGME). In this electrode, a fast response valve controls the drop growth. The cross sectional view of the CGME is shown in Figure 1.9 (BAS, 1993).

Figure 1.9

A diagram of the CGME

The capillary has a stainless stell tube embedded in the top end of the capillary. Mercury flow through the capillary is controlled by a fast response valve. The valve is rubber plug at the end of a shaft which when displaced slightly up will allow mercury to flow. Since the contact between filament of mercury in the capillary and the reservoir is a stainless steel tube, the electrode has low resistance. The total resistance from the contact point to the mercury is about 7 ohm. The valve seal is controlled by the valve seal adjustment knob. The opening of this valve is controlled by a computer-generated pulse sequence, which leads to a stepped increase in the drop size. Changing the number of pulse and/or the pulse width, so a wide range of drop sizes is available can therefore vary the drop size. Varying the time between the

39 pulses can control the rate of growth of the mercury drop. Therefore, a slowly growing mercury drop suitable for stripping experiments can be generated. An important advantage of the CGME compared to HMDE is no contamination of the capillary due to back diffusion (Dean, 1995). In addition to the mercury drop electrode, mercury may be deposited onto the surface of a solid electrode to produce mercury film electrode (MFE). The MFE is based on an electrochemically deposited mercury film on conventional substrate electrode such as a solid carbon, platinum, or gold electrode. The solid electrode is placed in solution of mercury ion (Hg2+) and held at a potential at which the reduction of Hg2+ to Hg is favorable, forming a thin mercury film. It displays the properties of a mercury electrode, having various electro analytical advantageous such as a high hydrogen evolution over potential and simple electrochemistry of many metals and other species of analytical interest. For example, Castro et al. (2004) have used thinfilm coated on a glassy carbon electrode (GCE) for quantitative determination of polycyclic aromatic hydrogen (PAH). In this experiment, they plated the mercury over 5 min at a cell voltage of -0.9 V. Economou and Fielden (1995) have developed a square wave adsorptive stripping voltammetric technique on the MFE for study of riboflavin. In this work, riboflavin was absorbed on the MFE at a potential of 0.0 V (vs. Ag/AgCl) in pH 12.0 of electrolyte solution. However, the increased risks associated with the use, manipulation and disposal of metallic mercury or mercury salt, have led to general trend for more environmentalfriendly analytical methods such as using bismuth film electrode (BFE) instead of mercury (Kefala et al., 2003; Economou, 2005; Lin et al., 2005a and Lin et al., 2005b ). The BFE consists of a thin bismuth film deposited on a carbon paste substrate that has shown to offer comparable performance to the MFE. Morfobos et al. (2004) have studied square wave adsorptive stripping voltammetry (SWAdSV) on a rotating-disc BFE for simultaneous determination of nickel (II) and cobalt (II). Hocevar et al. (2005) have developed a novel bismuth-modified carbon paste (Bi-CPE) for a convenient and

40 reliable electrochemical sensor for trace heavy metals detection in conjunction with stripping electroanalysis. In another study, using pencil-lead BFE as the working electrode, Demetriades et al. (2004) have determined trace metals by anodic stripping voltammetry (ASV).

They revealed that pencil-lead BFE were successfully applied to the determination of plumbum and zinc in tap water with results in satisfactory agreement with atomic absorption spectrometry (AAS). Zahir and Abd Ghani (1997) have developed a pencil 2B graphite paste electrode which was fabricated with polymerized 4-vinylpyridine for glucose monitoring. Other solid or metal electrode commonly used as WE are carbon, platinum (Salavagione et al., 2004; Santos and Machado, 2004; Aslanoglu and Ayne, 2004 ), gold (Hamilton and Ellis,1980; Parham and Zargar, 2001; Parlim and Zarger, 2003; Moressi et al., 2004; Munoz et al., 2005;), graphite (Orinakova et al., 2004; Pezzatini et al., 2004; Jin and Lin, 2005), diamond (Sonthalia et al., 2004) and silver (Iwamoto et al., 1984). Solid electrodes based on carbon are currently in widespread use in

voltammetric technique, primarily because of their broad potential window, low background current, rich surface chemistry, low cost, chemical inertness, and suitability for various sensing and detection application (Wang, 2000). It includes glassy carbon electrode (GCE), carbon paste electrode (CPE), chemically modified electrode (CME) and screen-printed electrode (SPE). For the GCE, the usual electrode construction is a rod of glassy carbon, sealed into an inert electrode body, a disc of electrode material is exposed to the solution. It is the most commonly used carbon electrode in electro analytical application (Ozkan and Uslu, 2002; Ibrahim et al., 2003; Erk, 2004). The cleaning of this electrode is important, to maintain a reactive and reproducible surface. Pre-treated electrochemically GCE have increased oxygen functionalities that contribute to more rapid electron transfer. Wang et al. (1997) have used in adsorptive potentiometric stripping analysis of tamoxifen, a nonsteriodal anti-estrogen used widely for the

41 treatment of hormone-dependent breast cancer. The electrode was anodised at + 1.7 V for 1 min in the electrolyte containing tomixifen. Using cyclic voltammetric technique, they found that a large definite anodic peak corresponding to the oxidation of the adsorbed drug at GCE at + 0.985 V. Other workers using GCE as the WE were Wang et al. (2004), Shi and Shiu (2004) and Ji et al. (2004).

The CPE, a mixture of carbon powder and a pasting medium at certain ratio, were the result of an attempt to produce electrode similar to the dropping mercury electrode (Kizek et al., 2005). They are particularly useful for anodic studies, modified electrode and also for stripping analysis. CMEs are electrodes which have been deliberately treated with some reagents, having desirable properties, so as to take on the properties of the reagents (Arrigan, 1994; Kutner et al., 1998). A few examples of these applications were reported by Abbaspour and Moosavi, (2002), Abbas and Mostafa (2003); Ciucu et al. (2003); Ferancova et al. (2000) and Padano and Rivas (2000). SPE are increasingly being used for inexpensive, reproducible and sensitive disposable electrochemical sensors for determination of trace levels of pollutant and toxic compounds in environmental and biological fluids sample (Wring et al., 1991). A disposable sensor has several advantages, such as preventing contamination between samples, constant sensitivity and high reproducibility of different printed sensors (Kim et al., 2002). The most useful materials for printing electrochemical sensors could be

carbon-based inks because they have a very low firing temperature (20-120° C) and can be printed on plastic substrate. Carbon can also be directly mixed with different compounds, such as mediator and enzymes. A few examples of the experiments which used SPE as the WE were reported by Kim et al. (2002), for detection of phenols using α-cyclodextrin modified screen printed graphite electrodes. Ohfuji et al. (2004) have constructed a glucose sensor based on a SPE and a novel mediator pyocyanin from Pseudomonas aeruginosa. Carpini et al. (2004) have studied oligonucleotide-modified

screen printed gold electrodes for enzyme-amplified sensing of nucleic acid. Lupu et al. (2004) have developed SPE for the detection of marker analytes during wine

42 making. In this work they have developed biosensors for malic acid and glucose with a limit of detection of 10-5 M and 10 -6 M for malic acid and glucose respectively. The sensors were applied in the analysis of different samples of wine. Besides using macro-electrode, voltammetric technique also utilizes microelectrode (Lafleur et al. 1990) with the size of electrode radius much smaller than the diffusion-layer thickness, typically between 7 to 10 µM (Hutton et al., 2005 and Buffle and Tercier-Waeber, 2005) as the WE. It is constructed from the same materials as the macro-electrode but with a smaller diameter to enhance mass transport of analyte to the electrode surface due to smaller electrode than the diffusion layer. Hence, increasing signal-to noise ratio and measurement can be made in highly resistive media due to decrease of the ohmic drop that results when the electrode size reduced (Andrieux et al., 1990). The microelectrode with diameter as small as 2 µm, allow voltammetric measurements to be made on even smaller sample (Harvey, 2002). Aoki (1990) reported that ultra microelectrodes influence electrode kinetics more specifically than conventional electrode because of lateral diffusion promotes mass transport. Since it minimise uncompensated resistance, they are useful for determination of kinetic parameters. Almeida et al. (1998) have done voltammetric studies of the oxidation of the anti-oxidant drug dipyridamole (DIP) in acetonitrile and ethanol using ultra microelectrode (UME). In this work they studied cyclic voltammetric technique of the drug with the UME which was 12.7 µm diameters. They found that with that electrode, the diffusion current of DIP is proportional to the electrode radii for low scan rates in cyclic voltammetry. The second electrode used in the voltammetric system is auxiliary electrode (AE). The AE is made of an inert conducting material typically a platinum electrode wire (Wang, 2000). It provides a surface for a redox reaction to balance the process that occured at the surface of the WE. It does not need special care, such as polishing. In order to support the current generated at the WE, the surface area of the AE must be equal to or larger than of the WE. The function of the AE is to complete the circuit, allowing charge flow through the cell (Fifield and Haines, 2000). In an electro

43 analytical experiment, there is no need to place the AE in a separate compartment since the diffusion of product that was produced by a redox reaction at the surface of the AE does not significantly interfere the redox reaction at the WE. The third electrode used in the voltammetric technique is reference electrode (RE). This RE provides a stable potential so that any change in cell potential is attributed to the working electrode. The major requirement for the RE is that the potential does not change during the recording voltammetric curve at different applied voltage (Heyrovsky and Zuman, 1968). The common RE are standard hydrogen electrode (SHE), calomel electrode (SCE) and silver/silver electrode (Ag/AgCl). The SHE is the reference electrode used to establish standard-state potential for other half reaction. It consists of a platinum electrode immersed in a solution in which the hydrogen ion activity is 1.00 and in which hydrogen gas is bubbled at a pressure of 1 atm. The standard-state potential for the reaction; 2H+ (aq)

+

2e-

→

H2 (g)

(1.0)

is 0.00 V for all temperatures. It is rarely used because it is difficult to prepare and inconvenient to use. The second reference electrode is the Standard Calomel Electrode (SCE) which is based on the redox couple between mercury chloride (Hg2Cl2) and Hg as below; Hg2Cl2(s) + 2e

2Hg(l)

+

2Cl-(aq)

(1.1)

The potential of this electrode is determined by the concentration of chloride ion. It is constructed using an aqueous solution saturated with potassium chloride (KCl) which has a potential of + 0.2444 V at 25 0 C. It consists of an inner tube packed with a paste of Hg, Hg2Cl2 and saturated KCl. A small hole connects the two tubes, an asbestos fiber serves as a salt bridge to the solution in which the SCE is immersed.

44 Other type of reference electrode is the Ag/AgCl electrode which is the most common RE since it can be used at higher temperature. This electrode is based on the redox couple between silver chloride ( AgCl ) and silver ( Ag ) as illustrated below;

AgCl(s)

+ 2e

Ag(s) +

Cl-

(1.2)

The potential of this electrode is determined by the concentration of Cl-. For saturated KCl the potential is + 0.197 V whereas for 3.5 M KCl the potential electrode is + 0.205 V at 25° C (Harvey, 2000). 1.3.2.2

Solvent and Supporting Electrolyte

Electrochemical measurements are commonly carried out in a medium which consists of solvent containing a supporting electrolyte. Sometimes in most cases, supporting electrolyte has to be added to the dissolved sample in an attempt to achieve the following (Heyrovsky and Zuman 1968); a) To make solution conductive b) To control the pH value so that organic substances are reduced in a given potential range and inorganic substances are not hydrolysed c) To ensure the formation of such complexes that give well developed and well separated waves d) To shift the hydrogen evaluation towards more negative potentials and to eliminate catalytic effects on hydrogen evolution e) To suppress unwanted maxima by addition of surface-active substances to the supporting electrolyte. The choice of the solvent is primarily by the solubility of the analyte, its redox activity and also by solvent properties such as electrical conductivity, electrochemical activity and chemical reactivity. The solvent should not react with the analyte and

45 should not undergo electrochemical reaction over a wide potential range. In aqueous solution the cathodic potential is limited by the reduction of hydrogen ions: 2 H+ (aq) + 2 e-

→

H2 (g)

(1.3)

resulting hydrogen evolution current. The more acidic the solution the more positive is the potential of this current due to the reaction expressed by; E

=

E0H2/H+ - 0.059 pH

(1.4)

The composition of the electrolyte may affect the selectivity of voltammetric measurements. The ideal electrolyte should give well-separated and well-shaped peaks for all the analytes sought, so that they can be determined simultaneously. For example Kontoyannis et al. (1999) used tris buffered saline (TBS) at pH 7.4 as the supporting electrolyte for simultaneous determination of diazepam and liposome using DPP technique. Inam and Somer (1998) have determined selenium (Se) and lead (Pb) simultaneously in whole blood sample by the same technique using 0.1 M HCl as the supporting electrolyte. They observed that there were three peaks at -0.33 V, -0.54 V and -0.41 V which belonged to an intermetallic compound (PbSe), Se and Pb respectively. Barbiera et al. (1997) have developed anodic stripping voltammetric technique for simultaneous determination of trace amounts of zinc, lead and copper in rum without pre-treatment and in the absence of supporting electrolyte. They observed that there were three peaks at -0.92 V, -0.42 V and 0.05 V which belong to Zn, Pb and Cu respectively. Because of the sensitivity of the voltammetric method, certain impurities in supporting electrolyte can affect the accuracy of the procedures. It is thus necessary to prepare the supporting electrolyte from highly purified reagents and should not easily oxidised and reduced. To obtain acceptable ionic strength of supporting electrolyte, certain concentration should be prepared which is usually about 0.1 M. This level is a compromise between high conductivity and minimum contamination (Wang, 2000).

46 The low ionic strength which is 0.01 M of supporting electrolyte (HClO4 – NaClO4) was very effective for the adsorptive accumulation of analyte on the electrode as found by Berzas et al. (2000) when they developed adsorptive stripping square wave technique for determination of sildenafil citrate (Viagra) in pharmaceutical tablet. Dissolved oxygen must be removed from supporting electrolyte first since the reduction of dissolved oxygen will cause two cathodic peaks at -0.05 V and -0.9 V (versus SCE) as reported by Fraga et al. (1998) and Reinke and Simon (2002). With increasing pH, the waves due to reduction of oxygen are shifted to more negative potential. The oxygen reduction generates a large background current, greater than that of the trace analyte, and dissolved oxygen therefore tends to interfere with voltammetric analysis (Colombo and van den Berg, 1998). The common method for the removal of dissolved oxygen is by purging with an inert gas such as nitrogen or argon where longer time may be required for large sample volume or for trace measurements. To prevent oxygen from reentering, the cell should be blanketed with the gas while the voltammogram is being recorded. However, this conventional procedure is time consuming and not suitable for flow analysis. Due to this reason, Colombo and van den Berg (1998) have introduced in-line deoxygenating for flow analysis with voltammetric detection. They have used an apparatus which is based on the permeation of oxygen through semi-permeable silicone tubing into an oxygen free chamber and enables the determination of trace metals by flow analysis with voltammetric determination. 1.3.2.3

Current in Voltammetry

When an analyte is oxidised at the working electrode, a current passes electrons through the external electric circuitry to the auxiliary electrode, where reduction of the solvent or other components of the solution matrix occurs. Reducing an analyte at the working electrode requires a source of electrons, generating a current that flows from the auxiliary electrode to the cathode. In either case, a current resulting from redox reaction at the working electrode and auxiliary electrode is called a faradaic current. A current due to the analyte’s reduction is called a cathodic current. Anodic currents are

47 due to oxidation reaction. The magnitude of the faradaic current is determined by the rate ofthe resulting oxidation or reduction reaction at the electrode surface. Two factors contribute to the rate of the electrochemical reaction which are the rate at which the reactants and products are transported to and from the electrode, and the rate at which electron pass between the electrode and the reactants and products in solution. There are three modes of mass transport that influence the rate at which reactant and products are transported to and from the electrode surface which are diffusion, migration and convection. Difussion from a region of high concentration to region of low concentration occurs whenever the concentration of an ion or molecule at the surface of electrode is different from that in bulk solution. When the potential applied to the WE is sufficient to reduce or oxidise the analyte at the electrode surface, a concentration gradient is established. The volume of solution in which the concentration gradient exists is called the diffusion layer. Without other modes of mass transport, the width of the diffusion layer increases with time as the concentration of reactants near electrode surface decreases. The contribution of diffusion to the rate of mass transport is time-dependent. Convection occurs when a mechanical means is used to carry reactants toward the electrode and to remove products from the electrode. Ther most common means of convection is to stirr the solution using a stir bar. Other methods include rotating the electrode and incorporating the electrode into a flow cell. Migration occurs when charged particles in solution are attracted or repelled from an electrode that has a positive or negative surface charge. When the electrode is positively charged, negatively charged particles move toward the electrode, while the positive charged particles move toward the bulk solution. Unlike diffusion and convection, migration only affects the mass transport of charged particles. The rate of mass transport is one factor influencing the current in voltammetry experiment. When electron transfer kinetics are fast, the redox reaction is equilibrium,

48 and the concentrations of reactants and products at the electrode are those psecified by Nersnt Equation. Such systems are considered electrochemically reversible. In other system, when electron transfer kinetics are sufficiently slow, the concentration of reactants and products at the electrode surface, and thus the current, differ from that predicted by the Nersnt euqation. In this case the system is electrochemically irreversible. Other currents that may exist in an electrochemical cell those are unrelated to any redox reaction are nonfaradaic and residual currents. The nonfaradaic current must be accounted for if the faradaic component of the measured current is to be determined. This current occurs whenever the electrode’s potential is changed. Another type of nonfaradaic current is charging current which occur in electrochemical cell due to the electrical double layer’s formation. Residual current is a small current that inevitably flows through electrochemical cell even in the absence of analyte (Harvey, 2000). 1.3.2.4

Quantitative and Qualitative Aspects of Voltammetry

Quantitative information is obtained by relating current to the concentration of analyte in the bulk solution and qualitative information is obtained from the voltammogram by extracting the standard-state potential for redox reaction. The concentration of the electroactive species can be quantitatively determined by the measurement of limiting current which is linear function of the concentration of electro active species in bulk solution. Half- potential serves as a characteristic of a particular species which undergoes reduction or oxidation process at the electrode surface in a given supporting electrolyte, and it is independent of the concentration of that species. Its function in the qualitative determination is the same as retention time in chromatographic technique.

49 1.3.3 1.3.3.1

Type of Voltammetric Techniques Polarography

Polarography is a subclass of voltammetry in which the WE is the DME. This technique has been widely used for the determination of many important reducible species since the DME has special properties particularly its renewable surface and wide cathodic potential range. In this technique, it takes place in an unstirred solution where a limiting current is diffusion limiting current (Harvey, 2000). Each new mercury drop grows in a solution whose composition is identical to that of the initial bulk solution. The relationship between the concentration of analyte, CA, and limiting current ( Id ) is given by Ilikovic equation ( Ewing, 1997; Heyrosky, 1968; Ilkovic, 1934 and Dahmen,1986). 2 1 3 6

1 2

I d = 708nD m t C A

(1.5)

where: n = number of electrons transferred in the redox reaction D = analyte’s diffusion coefficient ( cm2 sec-1) m = flow rate of mercury drop (g sec-1) t = drop time (sec) CA = concentration of depolariser (mol l-1) The above equation represents the current at the end of the drop life. The average current (Iave) over the drop life is obtained by integrating the current over this time period: 1

2

1

I ave = 607nD 2 m 3 t 6 C A

(1.6)

From the above equation, there is a linear relationship between diffusion current and concentration of electroactive species. It also indicates that the limiting diffusion

50 current is a function of the diffusion coefficient which depends on the size and shape of the diffusion particle. As compared to another technique such as cyclic voltammetry technique, in this technique, the peak current is directly proportional to concentration and increases with the square root of the scan rate as given by the Randles-Sevcik equation (Equation 1.7) for a reversible system (Wang, 2000). Ip = (2.69 x 105) n 3/2 ACD1/2υ1/2

(1.7)

where; n

=

number of electron

A

=

electrode area (cm2)

C

=

concentration (mol cm-3)

D

=

diffusion coefficient (cm2 s-1)

υ

=

scan rate (V s -1)

There are several types of polarographic techniques as was mentioned earlier. Polarography is used extensively in the analysis of metal ion, inorganic anions and organic compounds containing easily reducible or oxidisable functional group. A list of electroreducible and electrooxidisable organic functional groups is shown in Table 1.6 (Dean, 1995). Table 1.6

Electroreducible and electrooxidisable organic functional groups

Electroreducible organic functional groups

Aromatic carboxylic acid, azomethines, azoxy compounds conjugated alkene, conjugated aromatic, conjugated carboxylic acid conjugated halide, conjugated ketone, diazo compounds, dienes, conjugated double bond, nitroso compounds, organometallics, disulfide, heterocycles, hydroquinones, acetylene, acyl sulfide aldehyde, hydroxylamines, imines, ketones, nitrates, nitriles nitro compounds, oximes, peroxides, quinones, sulfones, sulfonium salts and thiocyanates.

Electrooxidisable organic functional groups

Alcohols, aliphatic halides, amines, aromatic amines, aromatic ides, carboxylic acids, ethers, heterocyclic amines, heterocyclic aromatics, olefins, organometallic and phenols.

51 1.3.3.2 Cyclic Voltammetry

Cyclic voltammetry (CV) is a potential-controlled reversal electrochemical experiment. A cyclic potential sweep is imposed on an electrode and the current response is observed (Gosser, 1993). CV is an extension of linear sweep voltammetry (LSV) in that the direction of the potential scan is reversed at the end of the first scan (the first switching potential) and the potential range is scanned again in the reverse direction. The experiment can be stopped at the final potential, or the potential can be scanned past this potential to the second switching potential, where the direction of the potential scan is again reversed. The potential can be cycled between the two switching potentials for several cycles before the experiment is ended at the final potential. CV is the most widely used technique for acquiring qualitative information about electrochemical reactions. Analysis of the current response can give considerable information about the thermodynamics of redox processes, the kinetics of heterogeneous electron-transfer reaction and the coupled chemical reactions or adsorption processes. It is often the first electrochemical experiment performed in an electrochemical study especially for any new analyte since it offers a rapid location of redox potentials of the electro active species and convenient evaluation of the effect of media upon the redox process. In the CV technique, the applied potential sweep backwards and forwards between two limits, the starting potential and the switching potentials. In this technique, the potential of the WE is increased linearly in an unstirred solution. The resulting plot of current versus potential is called a cyclic voltammogram. Figure 1.10a to 1.10c show the cyclic voltammograms for reversible, irreversible and quasireversible reactions. For a typical reduction and oxidation process in reversible reaction (as in Figure 1.10a), during the forward sweep the oxidised form is reduced, while on the reverse sweep the reduced form near the electrode is reoxidised. Chemical reaction coupled to the electrode reaction can drastically affect the shape of the CV response. In the case of irreversible reaction, no reverse peak is observed (Figure 1.10b).

52

(a)

(b)

(c) Figure 1.10

Cyclic voltammograms of (a) reversible, (b) irriverisble and (c)

quasireversible reactions at mercury electrode (O = oxidised form and R = reduced form)

53 The cyclic voltammogram shows characteristics of an analyte by several important parameters such as peak currents and peak potentials that can be used in the analysis of the cyclic voltammetric response either the reaction is reversible, irreversible or quasi-reversible as listed in Table 1.7. Cyclic voltammogram will guide the analyst to decide the potential range for the oxidation or reduction of the analyte and that it can be a very useful diagnostic tool. Table 1.7

Type of reaction

Reversible

The characteristics of different type of electrochemical reaction

Characteristics

Cathodic and anodic peak potential are separated by 59/n mV. The position of peak voltage do not alter as a function of voltage scan rate The ratio of the peak current is equal to one The peak currents are proportional to square root of the scan rate The anodic and cathodic peaks are independent of the scan rate Disappearance of a reverse peak

Irreversible The shift of the peak potential with scan rate ( 20 – 100 mV/s) Peak current is lower than that obtained by reversible reaction. Quasi-reversible

Larger separation of cathodic and anodic peak potential (> 57/ n mV) compared to those of reversible system.

54 1.3.3.3

Stripping Voltammetry

Stripping technique is one of the most important and sensitive electrochemical technique for measuring trace metals and organic samples. The term stripping is applied to a group of procedures involving preconcentration of the determinant onto the working electrode, prior to its direct or indirect determination by means of a potential sweep (Wang, 1985). The preconcentration (or accumulation) step can be adsorptive, cathodic or anodic. Its remarkable sensitivity is attributed to the addition of an effective preconcentration step with advanced measurement procedures that generate an extremely favorable signal-to-noise ratio as reported by Blanc et al. (2000). Brainina et al. (2000) list advantages of this technique such as high sensitivity, low detection limit, wide spectrum of test materials and analytes, both of organic and inorganic origin, insignificant effect of matrix in certain instances, compatibility with other methods, relative simplicity and low cost of equipment and finally, it can be automatic on-line and portable options. This technique is able to measure many analytes simultaneously such as reported by Ghoneim et al. (2000). They have determined up to eleven metals in water sample simultaneously using this technique. Stripping voltammetry technique utilises a few steps as follows; a)

Deposition step: In this step, analyte will be preconcentrated on the WE within a certain time

while solution is stirred. The deposition potential imposed on the WE is chosen according to the species to be determined and is maintained for a deposition period depending on their concentration. The choice of deposition potential can provide some selectivity in the measurement (Sun et al., 2005). Deposition time must be controlled since the longer the deposition time, the larger the amount of analye available at the electrode during stripping. During deposition step, the solution is stirred to facilitate transportation of ions of interest to the surface of WE.

55 b)

Rest step: In this step, it allows formation of a uniform concentration of the ions of interest

on the mercury. As the forced convection is stopped at the end of the deposition period, the deposition current drops almost to zero and a uniform concentration is established very rapidly. It also insures that the subsequent stripping step is performed in a quiescent solution. c)

Stripping step: This step consists of scanning the potential anodically for anodic stripping and

cathodically for cathodic stripping. When the potential reaches the standard potential of a certain ion of interest–metal ion complex, the particular ion of interest is reoxidised or reduced back into solution and a current is flowing. The resultant voltammogram recorded during this step provide the analytical information of the ions of interest. The stripping step after preconcentration gives the possibility for selective determination of different substances assuming that they also have different peak potential as reported by Kubiak et al. (2001). The potential-time sequence in stripping analysis is shown in Figure 1.11 which shows all three steps that were mentioned earlier.

Applied Potential

Deposition

Stripping

Stirred

Quiescent

Deposition potential

Final potential

Time

Figure 1.11

The potential-time sequence in stripping analysis

56 Most stripping measurements require the addition of appropriate supporting electrolyte and removal of dissolved oxygen. The former is needed to decrease the resistance of the solution and to ensure that the metal ions of interest are transported toward the electrode by diffusion and not by electrical migration. Contamination of the sample by impurities in the reagents used for preparing the supporting electrolyte is a serious problem. Dissolved oxygen severely hampers the quantitation and must be removed. The main types of interference in stripping analysis are overlapping stripping signals, the adsorption of organic surfactants on the electrode surface, the presence of dissolved oxygen and the formation of intermetallic compounds by metals such as copper and zinc co-deposited in the mercury electrode. Overlapping signals cause problems in the simultaneous determination of analytes with similar redox potential such as lead and tin. Intermetallic-compounds formation and surfactant cause a depression of the stripping response and also shifting the signal location. Stripping voltammetry is composed of three related techniques that are anodic, cathodic and adsorptive stripping voltammetry. 1.3.3.3a

Anodic Stripping Voltammetry (ASV)

ASV is mainly used in the determination of metal ions that can be reduced and then re-oxidised at a mercury electrode. Voltammetric measurements of numerous metal ions in various types of samples have been reported (Arancibia et al., (2004) and Shams et al., (2004)). The term ASV is applied to the technique in which metal ions are accumulated by reduction at an HMDE held at a suitable negative potential. The deposition potential is usually 0.3 to 0.5 V more negative than a standard potential for reduced metal ion to be determined. ASV consists of two steps. The first step is a controlled potential electrolysis in which the working electrode is held at a cathodic potential sufficient to deposit the metal ion (M n+) on the electrode to form an amalgam, M(Hg). This step is called accumulation step which is represented by an equation;

57 Mn+ +

ne-

+

Hg → M(Hg)

(1.8)

The solution is stirred during this process to increase the rate of deposition. Near the end of the deposition time, stirring is stopped to eliminate convection as a mode of mass transport. The duration of the deposition step is selected according to the concentration level of the metals ion. In the second step, the potential is scanned anodically toward more positive potential. When the potential of the WE is sufficiently positive the analyte is stripped from the electrode, returning to solution as its oxidised form. This step is called stripping which is represented by an equation M(Hg) → Mn+

+

ne-

(1.9)

The current during the stripping step is monitored as a function of potential giving rise to a peak-shaped voltammogram. The peak current is proportional to the analyte’s concentration. 1.3.3.3b

Cathodic Stripping Voltammetry (CSV)

CSV is used to determine a wide range of organic compounds, and also inorganic compounds that form insoluble salts with the electrode material. It has been found to be widely applicable to many problems of clinical and pharmaceutical interest (Wang, 1988). Voltammetric measurements of numerous electro active species of biological significance such as drugs (Ghoneim et al., 2003; Arranz et al., 1999; Rodriguez et al., 2004; Ghoneim and Beltagi, 2003 and Cabanillas et al., 2003 ) and toxic substances ( Hourch et al., 2003; Safavi et al., 2004 ) have been reported. The term CSV was used originally for the indirect trace determination of organic compounds as mercury salt, involving anodic oxidation of mercury and subsequently cathodic reduction of mercury. This technique is similar to the previous

58 technique with two exceptions. First, the deposition step involves the oxidation of the mercury electrode from Hg to Hg2+, which then reacts with the analyte to form an insoluble salt at the surface of the electrode. For example, when chloride ion (Cl-) is the analyte, the deposition step is 2Hg(l) +

2Cl- (aq)

→

Hg 2Cl2 (s) + 2e-

(1.10)

Secondly, stripping is accomplished by scanning cathodically toward a more negative potential, reducing Hg2+ back to Hg and returning the analyte to the solution. Hg2Cl2(s) + 2e- → 1.3.3.3c

2Hg(l) + 2Cl-(aq)

(1.11)

Adsorptive Stripping Voltammetry (AdSV)

The term AdSV seems to have been first used by Lam et al. in 1983. It is a powerful analytical technique for the determination of nmol levels of a wide range of organic compounds (Smyth and Smyth, 1978; Smyth and Vos, 1992). In technical report for International Union Of Pure And Applied Chemistry under Analytical Chemistry Division Commission On Electro analytical Chemistry (Fogg and Wang 1999), suggested that AdSV technique is applied to stripping voltammetric technique in which accumulation is effected by adsorption of mainly organic determinants and that can be justified less in case of the adsorption of metal complexes in determining metal ions. The AdSV term should not be applied when there is a change of oxidation state of the metal ion during the accumulation for example in the accumulation of copper (I) complexes or salts or in other cases where an organic compounds is being accumulated, and determined indirectly, as a metal salts or complex such as mercury salts or nickel complexes. In this technique the deposition step occurs without electrolysis process. Instead, the analyte adsorbs on to the electrode’s surface. During deposition, the electrode is maintained at a potential that enhances adsorption. When deposition is

59 sufficient the potential is scanned in an anodic or cathodic direction depending on whether we wish to oxidise or reduce the analyte. In recent years, AdSV which improves sensitivity and selectivity, have promoted the development of many electrochemical methods for ultra-trace measurement of a variety of organic (Ghoneim et al., 2003; Ghoniem and Taufik, 2004; Farias et al., 2003 and Hourch et al., 2003) and inorganic species (Ensafi et al., 2004; Zimmerman et al., 2001) and JuradoGonzalez et al. (2003). Barek et al. (2001a) reported that AdSV on HMDE is much more sensitive with a typical LOD between 10-9 and 10 -10 M. 1.3.3.4

Pulse voltammetry

This technique uses pulse waveform in recording its voltammogram which offers enhanced sensitivity and resolution. The advantage of pulse techniques is that the waveform is designed so as to discriminate against non-faradic current hence, increase sensitivity. The enhanced resolution is particularly useful when several electroactive species are being analysed simultaneously (Fifield and Haines, 2000). In this technique, current sampling takes place at the end of the pulse and utilise the different time dependence of faradic (if) and charging current (ic), as shown in Figure 1.12.

Faradaic Current, i f

+

Sampling time

Changing Current, i c

Current

0

Figure 1.12

Pulse time

Time

Schematic drawing showing the if and ic versus pulse time course

60 This technique, is aimed at lowering the detection limits of voltammetric measurement down to 10-8 M in its differential pulse mode. Increasing the ratio between the faradic and non-faradic current permit convenient quantitation down to the 10-8 M concentration level (Wang, 2000). Differential pulse and square wave techniques are the most commonly used pulse technique. Differential pulse polarograms or voltammograms are peak shaped because current difference is measured. The following section gives an overview of three important waveforms in pulse technique. 1.3.3.4a

Differential Pulse Voltammetry (DPV)

In DPV, fixed magnitude pulse (10 to 100 mV) superimposed on a linear potential ramp are applied to the working electrode at a time just before the end of the drop as shown in Figure 1.13. The current is sampled twice, just before the pulse application and again late in the pulse life normally after 40 ms, when the charging current has decayed. Subtraction of the first current sampled from the second provides a stepped peak-shape derivative voltammogram. The resulting differential pulse voltammogram consists of current peaks, the height of which is directly proportional to the concentration of corresponding analyte. The differential-pulse operation results in a very effective correction of the charging background current.

Pulse Width Step E

E

Sample Period

Pulse Amplitude Sample Period Pulse Period

Outlet Time t

Figure 1.13

The schematic diagram of steps in DPV by superimposing a periodic

pulse on a linear scan

61 1.3.3.4b

Square Wave Voltammetry (SWV)

SWV is a large-amplitude differential technique in which a waveform composed of a symmetrical square wave, superimposed on a base staircase potential, is applied to the working electrode. The current is sampled twice during each squarewave cycle, once at the end of forward pulse and another at the end of the reverse pulse. Since the square-wave modulation amplitude is very large, the reverse pulses cause the reverse reaction of the product of the forward pulse. The difference between the two measurements is plotted versus the base staircase potential as shown in Figure 1.14. The resulting peak current is proportional to the concentration of the analyte. Excellent sensitivity accrues from the fact that the net current is larger than either the forward or reverse components. Coupled with the effective discrimination against the charging current, very low detection limits can be attained.

1 ESW

∆E

2

Applied Potential

τ

Accumulation Time Time

Figure 1.14

Waveform for square-wave voltammetry

The advantage of SWV is that a response can be found at a high effective scan rate, thus reducing the scan time. Because of its high scan rate, it provides a great economy of time (Arranz et al., 1999 and Ghoneim and Tawfik., 2004). There are

62 several reports on application of the SWV technique for determination of several samples as listed in Table 1.8 which involves deoxygenation step prior analysis. However, in certain case beside it offers the additional advantage of high speed; it can increase analytical sensitivity and relative insensitivity to the presence of dissolved oxygen as reported by Economou et al. (2002). Table 1.8

Application of SWV technique

No

Sample

Supporting electrolyte

Reference

1

Ketorolac in human serum

Acetate buffer, pH 5.0

Radi et al. (2001)

2

Imidacloprid in river water

BRB, pH 7.2

Guiberteau et al. (2001)

3

Cocaine and its metabolite

Phosphate buffer, pH 8.5

Pavlova et al. (2004)

4

Amlodipine besylate in BRB, pH 11.0 tablets and biological fluids

Gazy (2004)

5

EDTA species in water

Diluted HCl, pH 2.8 and 0.05 M NaCl

Zhao et al. (2003)

6

RDX in soil

0.1 M acetate buffer, pH 4.5

Ly et al. (2002)

7

Levofloxacin in human urine

0.05 M acetate buffer, pH Radi and El-Sherif 5.0 (2002)

8

Sertraline in commercial products

0.1 M borate, pH 8.2

Nouws et al. (2005a)

63 Table 1.8

No

Sample

continued

Supporting electrolyte

Reference

9

Cadmium in human hair

0.01 M TEA, pH 11.0

Arancibia et al. (2004)

10

Copper, stannous, antimony, thallium, and plumbum in food and environmental matrices

0.1 M dibasic ammonium Locatelli (2005) citrate, pH 6.3

11

Imatinib (Gleevec) and its metabolite in urine

0.012 M HClO4, pH 2.0

12

Famotidine in urine

0.02 M MOPS buffer, pH Skrzypek et al. 6.7 (2005)

13

Haloperidol in bulk form, BRB, pH 9.0 pharmaceutical formulation and biological fluids

El-Desoky et al. (2005)

14

Copper, cadmium and zinc complexes with cephalosporin antibiotic

Acetic acid, pH 7.34

El-Maali et al. (2004)

15

Triprolidine in pharmaceutical tablets

0.04 M BRB, pH 11.0

Zayed and Habib (2005)

16

Metoclopramide in tablet and urine

0.4 M HCl-sodium Farghaly et al. acetate, pH 6.2 and 0.2 M (2005) KCl

17

Cefoperazone in bacterial culture, milk and urine

BRB, pH 4.4

Rodriguez et al. (2005)

Billova et al. (2005)

64

Table 1.8

continued

Notes:

BRB: EDTA: HClO4: MOPS: RDX: TEA:

Britton-Robinson buffer Ethylene diamine tetraacetic acid Perchloric acid 3-(N-morpholino)propanesulphonic Hexahydro-1,3,5-trinitro-1,3,5-triazine Triethylammonium

1.4

Objective and Scope of Study

1.4.1

Objective of Study

The development of methods for the determination of aflatoxins has been constantly in demand due to the fact that aflatoxins are a major concern as the toxic contaminants of foodstuffs and animal feed, and have been recognised as a potential threat to human health since the early 1960s resulting in frequent economic losses. The widespread occurrence of aflatoxins producing fungi in our environment and the reported natural occurrence of toxin in a number of agricultural commodities has led the investigator to develop a new method for aflatoxins analysis. In order to maintain an effective control of aflatoxins in food and foodstuffs proper analytical procedures must be applied. Different analytical methods such as thin layer, liquid chromatography or enzyme immunoassay test which were mentioned in Chapter I, have been developed for aflatoxins determination. However all these proposed techniques have their disadvantages. For HPLC technique, the method requires well equipped laboratories, trained personnel, harmful solvents as well as time consuming, and costly in buying and maintenance.

65 For immunological methods such as ELISA technique, it has a few disadvantages such as long incubation time, washing and mixing steps and also labour intensive. This method requires highly specific polyclonal or monoclonal sera which is costly. For radioimmunoassay (RIA) technique, it uses radioisotope which raises concerns in radiation safety in dealing with and also in disposing radioactive waste. For micellar electrokinetic capillary chromatography (MECC) technique, it needs preparing a lot of reagents for the buffering system and takes considerable time to complete the analysis. With regards to voltammetric analysis, no stripping voltammetric study of aflatoxins is reported until now. This study has been proposed in order to develop a new alternative technique for determination of aflatoxins. This technique is the most promising method for determination of aflatoxins due to its main advantages compared to competing analytical techniques such as excellent sensitivity, reasonable speed, minimal sample pre-treatment, satisfactory selectivity, wide applicability, ability to undertake speciation analysis and low cost of instrumentation and maintenance as reported by Economou et al. (2002) and Radi (2003). Stripping analysis has been proven to be a powerful technique for the determination of both organic and inorganic electroactive species (Bond, 1980). Differential pulse cathodic stripping voltammetry satisfies the requirement of an efficient and senstitive technique for the determination of aflatoxin compounds because it has been shown to be a powerful technique in determination of other organic compounds in various sample origins down to ppb level as reported by Zima et al. (2001), Sun and Jiao (2002), Yardimer and Ozaltin (2004) and Nouws et al. (2005b). In this research, the voltammetric behaviour of these compounds would be studied in great detail and the stripping voltammetry especially differential pulse stripping voltammetry (DPSV) and square wave stripping voltammetry (SWSV) techniques could provide accurate and sensitive methods for aflatoxins determination especially in food sample such as groundnuts, would be investigated.

66 The groundnut is selected for the sample to be analysed in this study since among foods and foodstuff, peanut and peanuts products are widely utilized as health food (www.copdockmill.co.uk/aflatoxin). It seem to be significantly contaminated with aflatoxins. It contains high content of carbohydrate which provides a substrate that is particularly suitable for toxin production (Neal, 1987). This will affect the quality and safety of humans life. Futhermore, Aspergillus moulds which produces aflatoxins grow easy on groundnut. AFB1, AFB2, AFG1 and AFG2 were selected as the subjects in this study due to the regulatory requirement for their determination in imported raw grounnuts as imposed by the Malaysian government to assure that this commodity is free from any toxin contamination before being supplied for the domestic market. AFM1 and AFM2 are not involved in this study since currently no such regulation imposed by the Malaysian government even though a regulatory standard has already been set which is not more 0.05 ppb AFM1 and AFM2 should be present in milk and milk products. For the time being, the analysis on AFM1 and AFM2 are not being carried out in Malaysia. The other reason, globally, AFB1, AFB2, AFG1 and AFG2 are more concerned with their contamination in many food samples compared to AFM1 and AFM2 which contaminate milk and milk products only. The objectives of this study are: a)

To investigate the electrochemical behaviour of aflatoxins B1 (AFB1), AFB2, AFG1 and AFG2 on the mercury electrode in appropriate supporting electrolyte.

b)

To establish optimum conditions for the determination of those aflatoxins by the method of differential pulse cathodic stripping voltammetry (DPCSV) and square wave stripping voltammetry (SWV) techniques with a HMDE as the working electrode using Britton-Robinson buffer (BRB) solution as the supporting electrolyte.

67 c)

To develop an accurate, sensitive, fast and simple method for the determination of all studied aflatoxins in food samples such as ground nut and comparing the results with the established method such as HPLC.

1.4.2 Scope of Study

The studies are as follows: a)

Studies on the voltammetric behaviour of aflatoxin compounds using cyclic voltammetry (CV) technique as an introductory step. Using this technique, the effect of increasing concentration of aflatoxins, scan rate and repetitive scanning on the peak height (Ip) and peak potential (Ep) of each aflatoxin will be investigated.

b)

Studies on the differential pulse cathodic and anodic stripping voltammetriy (DPCSV) of all aflatoxins. Parameters optimisation include pH of supporting electrolyte, accumulation potential (Eacc), accumulation time (tacc), scan rate (υ), initial potential (Ei), final potential (Ef) and pulse amplitude.

c)

Using optimised analytical parameters and experimental conditions, the effect of increasing concentration of aflatoxins to the Ip of the compounds will be studied. Regression equation, R2 value, linearity range, limit of detection (LOD), limit of quantification (LOQ), accuracy and reliability of the method will be obtained. Ruggedness and robustness tests also will be studied for the proposed technique.

d)

The proposed technique will be further investigated in terms of interference where each aflatoxin will be reacted with increasing amounts of metals ion such as zinc, aluminum, nickel, lead and copper, and with organic compounds such as ascorbic acid, L-cysteine and β-cyclodextrin.

68 e)

The optimised parameters for DPCSV technique will be applied for square wave stripping voltammetry (SWSV) including optimisation steps such as the Eacc, tacc, pulse amplitude, scan rate, voltage step and frequency. The last 3 parameters are interrelated to each other in the SWSV technique.

f)

Both DPCSV and SWSV techniques that were successfully developed will be applied in the determination of aflatoxins content in real samples such as groundnut. The recovery studies also will be carried out for the accuracy test of the developed method. The results will be compared with that obtained by accepted technique such HPLC. For the HPLC analysis, the final solutions from the extraction and clean-up procedures of groundnut in chloroform were sent to the Chemistry Department, Penang Branch, Ministry of Science, Technology and Innovation (MOSTI).

g)

The stability of aflatoxins will be determined for aflatoxin stock and standard solutions according to the following procedures i)

Aflatoxin stock solutions prepared in benzene: acetonitrile (98:2) will be studied using ultra-violet-visible spectrophotometer. In this analysis, each aflatoxin stock solution will be monitored every month (from 0 to 12 months) and the concentrations of aflatoxins will be calculated from the measured absorbance.

ii)

Aflatoxin standard solutions in BRB which was kept in the freezer at -4.0 0 C will be measured using voltammetric technique monthly up to 6 months

iii)

Aflatoxin standard solutions in BRB which have been added into the voltammetric cell and exposed to ambient temperature will be onitored every hour (from 0 to 8 hours) by measuring their peak height and peak potential

69 iv)

Aflatoxin standard solution in BRB which was added into the voltammetric cell containing BRB at different pH (6.0, 7.0, 9.0 and 11.0) and exposed to ambient temperature will be monitored every hour (from 0 to 3 hours) by measuring their peak height and peak potential.

70

CHAPTER II

RESEARCH METHODOLOGY

2.1

Apparatus, Materials and Reagents

2.1.1

Apparatus

A BAS CV-50W voltammetric analyser in connection with a Control Growth Mercury Electrode (CGME) stand equipped with a three-electrode system and a 20 ml capacity BAS MR-1208 cell as shown in Figure 2.0 were used for all the voltammetric determinations. The working electrode was a Hanging Mercury Drop Electrode (HMDE). As a reference and counter-electrode, a silver-silver chloride (Ag / AgCl) and a platinum wire were used, respectively. All potentials are quoted relative to this reference electrode. BAS CV-50W was connected to a computer for data processing. For robustness test which is required in validating procedure for developed technique, VA 757 Computrace Metrohm Voltammetric analyzer as shown in Figure 2.1 was used. A pH meter Cyber-scan model equipped with a glass electrode combined with an Ag/AgCl reference electrode, was employed for all pH measurements. UV-VIS spectrophotometer (UV-2501/PC Shimadzu) was used for measurement of absorbance of all 10 ppm aflatoxin stock solutions and 1 ppm aflatoxin standard in BRB solution.

71

(a)

(b)

Figure 2.0 BAS CGME stand (a) which is connected to CV-50W voltammetric analyser and interface with computer (b) for data processing

Figure 2.1 VA757 Computrace Metrohm voltammetric analyzer with 663 VA Stand (consists of Multi Mode Electrode (MME))

72

2.1.2

Materials

2.1.2.1 Aflatoxin Stock and Standard Solutions

Aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 ( AFG2) (SIGMA) are supplied by Chemopharm with a quantity of 1 mg per bottle each. The batch numbers of these aflatoxins are listed in Table 2.0. Table 2.0

List of aflatoxins and their batch numbers used in this experiment.

Aflatoxins

Batch Number

AFB1

112K4049

AFB2

120K4014

AFG1

11K4003

AFG2

082K4091

Stock solutions of these aflatoxins with concentrations of 10 ppm each were prepared by dissolving the total amount of aflatoxin with 2.0 ml acetonitrile. The solution was transfered into 100 ml volumetric flask. Benzene was added into the flask until 100 ml mark. The flasks were covered with aluminium foil and kept in refrigerator with a temperature of 16° C. The absorbances of these stock solutions were measured using ultraviolet-visible (UV) spectrometer and the concentrations of aflatoxins were calculated. This measurement of these aflatoxins stock solutions were carried out monthly per a year while kept under cool condition for stability study.

73 For preparation of aflatoxins standard solution with concentration of 1 ppm, 1 ml of the stock solution was pipetted and placed into an amber bottle. Nitrogen was bubbled into the solution to remove all the organic solvent until dryness followed with an addition of 10 ml BRB at pH of 9.0. The solution was well mixed and kept in freezer at temperature of -4 0 C. This standard solution was analysed by voltammetric technique very month for 6 months to observe the stability of the aflatoxins in buffered solution. To obtain the concentration of 0.1 µM of AFB1, AFB2, AFG1 and AFG2 each in 10 ml supporting electrolyte, an amount of 312, 315, 328 and 330 µl was injected into the cell respectively.

2.1.2.2

Real Samples

Real samples such as groundnut were purchased from a few domestic shops which are located in Taman Universiti, Skudai, Johor. These samples were labeled as S01 to S15. 2.1.3

Reagents

The chemicals used were all of analytical reagent quality grade and the solutions were prepared with de-ionised water obtained from a Barnstead Ultra Nanopure water system. 1% of sodium hypochlorite was used to clean all glasswares used. 2.1.3.1

Britton-Robinson Buffer (BRB), 0.04 M

BRB with a buffering range from 2.0 to 12 was prepared by dissolving 2.47 g of boric acid (Fluka) in a 1liter flask containing 2.3 ml of glacial acetic acid (MERCK) and 2.70 ml orthophosphoric acid (Ashland Chemical) and then further diluting to the 1 liter mark with deionised water. The pH of the buffer solution was adjusted to the

74 required value by addition of 1.0 M sodium hydroxide (MERCK) or 1.0 M hydrochloric acid solutions (MERCK). 2.1.3.2

Carbonate Buffer, 0.04 M

Carbonate buffer with a buffering range from pH 9.0 to 12 was prepared by mixing two different solutions which were firstly prepared. The first solution, 0.04 M sodium bicarbonate was prepared by dissolving 3.36 g sodium bicarbonate (MERCK) in a 1 liter of de-ionised water. Similarly, for the second solution, 0.04 M sodium carbonate was prepared by dissolving 4.24 g sodium carbonate (MERCK) in 1 liter of deionised water. The pH of the buffer was adjusted to the required value by mixing the two solutions. 2.1.3.3

Phosphate Buffer, 0.04 M

Phosphate buffer was prepared by mixing two different solutions which were firstly prepared. The first solution, 0.04 M potassium hydrogen phosphate was prepared by dissolving 5.44 g potassium hydrogen phosphate (BDH) in a 1 liter of deionised water. Similarly, for the second solution, 0.04 M sodium hydrogen phosphate was prepared by dissolving 5.68 g sodium hydrogen phosphate (BDH) in 1 liter of deionised water. The pH of the buffer was adjusted to the required value by mixing the two solutions. 2.1.3.4

Ascorbic Acid Solution, 1.0 mM

This solution was prepared by dissolving 0.0176 g of ascorbic acid (GCE) in 100 ml of de-ionised water.

75 2.1.3.5

β-cyclodextrin (β-CD) Solution, 1.0 mM

This solution was prepared by dissolving 0.1135 g of C42H70O35 (Fluka) in 100 ml of de-ionised water. L-cysteine, 1.0 x 10-5 M

2.1.3.6

This solution was prepared by dissolving 0.012 g of cysteine (SIGMA) in 100 ml of de-ionised water. 1.0 ml of this solution was diluted to 100 ml with de-ionised water. 2.1.3.7

2,4-dihydrofuran, 0.15 M

This solution was prepared by diluting 1.105 ml of 13.57 M of 2,4 dihydrofuran (SIGMA) into 100 ml with de-ionised water. Coumarin, 3.0 x 10 -2 M

2.1.3.8

This solution was prepared by dissolving 0.21 g of coumarin (SIGMA) in 50 ml methanol. 2.1.3.9

Poly-L-lysine (PLL), 10 ppm

This solution was prepared by dissolving 0.001 g of poly-l-lysine (SIGMA) in 100 ml de-ionised water. 2.1.3.10

Standard Aluminium (III) Solution, 1.0 mM

The standard aluminium (III) solution was prepared by dissolving 0.5212 g of Al(NO 3)3.9H2O (MERCK) in 100 ml de-ionised water.

76 2.1.3.11

Standard Plumbum (II) Solution, 1.0 mM

The standard plumbum (II) solution was prepared by dissolving 0.0530 g Pb(NO3)2 (EMORY) in 100 ml deionised water. 2.1.3.12

Standard Zinc (II) Solution, 1.0 mM

The standard zinc (II) solution was prepared by dissolving 0.121g ZnSO4.5H2O (BDH) in 100 ml de-ionised water. 2.1.3.13

Standard Copper (II) Solution, 1.0 mM

The standard copper (II) solution was prepared by dissolving 0.0980 g. CuSO4.7H2O (GCE) in 100 ml de-ionised water. 2.1.3.14

Standard Nickel (II) Solution, 1.0 mM

The standard nickel (II) solution was prepared by dissolving 0.0963 g NiCl2.6H2O (MERCK) in 100 ml deionised water. 2.1.3.15

Methanol: 0.1 N HCl Solution, 98 %

This solution was prepared by mixing 98 ml of methanol and 2 ml 0.1 N HCl. 2.1.3.16

Zinc Sulphate Solution, 15%

This solution was prepared by dissolving 15 g of zinc sulphate (MERCK) in 100 ml de-ionised water.

77 2.2

Analytical Technique

2.2.1

General Procedure for Voltammetry Analysis

The general procedure used to obtain stripping voltammograms was according to the following procedures. A 10 ml aliquot of buffer solution was placed in a voltammographic cell and the required aflatoxin standard solution was added using a micropipette. The stirrer was switched on at 300 rpm and the solution was purged with nitrogen gas for 10 minute. After forming a new HMDE, accumulation was effected for the required time at the appropriate potential while stirring the solution. A medium mercury drop size was used. At the end of the accumulation time the stirring was switched off and after 10 s had elapsed to allow the solution to become quiescent, the negative going potential was initiated. When further volume of aflatoxin were added to the cell, the solution was redeoxygenated with nitrogen gas for another 2 minute before carrying out further voltammetric determination following the mentioned general procedures. 2.2.2

Cyclic Voltammetry (Anodic and Cathodic Directions)

Cyclic voltammetry of all aflatoxins were carried out by anodic and cathodic cyclic techniques. The initial parameters for cathodic cyclic voltammetry are as follows; initial potential (Ei) = 0 V, high potential, (EH) = 0 V, low potential (EL) = 1.50 V, scan rate (υ)= 200 mV/s, quiet time = 10 s and sensitivity = 1 µA/V. For anodic cyclic voltammetry, all previous parameters were maintained except for Ei = -1.50 V. The concentration of aflatoxins used for these experiments were fixed at 1.3 µM. 2.2.2.1

Standard Addition of Sample

5 ppm (1.59 x 10-5 M) AFB2 standard solution was prepared by taking 5.0 ml aflatoxin stock solution, degassed until dryness and redissolved in BRB pH 9.0. For cyclic voltammetry experiment, general procedure as previouly mentioned was

78 followed. A 818 µl of 1.59 x 10-5 M AFB2 standard solution was then added into voltammetric cell containing 10 ml of supporting electrolyte which resulted in concentration of aflatoxin of 1.3 µM in the cell. The solution was purged with nitrogen for 120 s before being scanned. Further series of scans were carried out with increasing concentration of the aflatoxins from 2.0 to 3.4 µM. Subsequently a purge of 120 s was employed between the cyclic voltammetry. A graph of peak current against aflatoxin concentration was plotted and linearity of the curve was calculated. This method was repeated for other aflatoxins. 2.2.2.2

Repetitive Cyclic Voltammetry

Using the aflatoxins with concentration of 1.3 µM, repetitive cyclic voltammetry for anodic and cathodic direction were run using the same parameters as mentioned before with the υ of 200 mV/s and 10 cyclic number. Any change in the Ip and Ep height of all aflatoxins due to the increasing number of the cycle were observed. 2.2.2.3

Effect of Scan Rate (υ)

The whole procedure for cyclic voltammetry was repeated for all aflatoxins with different υ from 20 mV/s to 500 mV/s while other parameters kept constant. The applied υ were 20, 40, 60, 80, 100, 200, 300, 400 and 500 mV/s. Any change in the Ip and Ep of all aflatoxins due to the changing of the υ were observed. Graphs of log Ip against log υ, Ep versus log υ and Ip versus υ were plotted. 2.2.3

Differential Pulse Cathodic Stripping Voltammetric Determination of AFB2

A 315 µl of 1 ppm (0.318 x 1-0-5 M) AFB2 sample solution in BRB at pH 9.0 was added by means of micropipette into the cell which contained 10 ml of the BRB with the same pH. The resulting concentration of sample in the voltammetric cell is 0.1 µM (31.4 ppb). The solution was deoxygenated with nitrogen free oxygen for 120 s

79 before carrying out further voltammetry. Three further series of sample additions were employed with a purge of 120 s were performed between each cathodic stripping cycle. 2.2.3.1

Effect of pH

The following general procedure was carried out. A 1.26 ml of 5 ppm (1.59 x -5

10 M) AFB2 standard solution was added by means of micropipette into the cell which contained 8.74 ml of BRB at pH 9.0. The resulting concentration of sample in voltammetric cell is 2.0 µM (628 ppb). The solution was deoxygenated with nitrogen for 120 s before carrying out further voltammetry. The whole procedure was repeated for series of buffer from pH 4.0 to 13.0 and a graph of Ip vs. pH was then plotted. 2.2.3.2

Method Optimisation for the Determination of AFB2

The optimisation steps for determination of AFB2 were carried out using two different concentrations of AFB2 which were 2.0 uM and 0.02 µM for high and low concentrations respectively. The initial parameters used for this optimisation steps are; Ei = 0 mV, Ef = -1500 mV, Eacc = 0 mV, t acc = 0, υ = 20 mV/s, pulse implitude = 100 mV and quite time = 10 s. General procedure for voltammetric analysis was employed. The Ip and Ep values were observed. The same procedure was repeated with the same parameters except for tacc was 30 s. For the optimisation steps, the influence of each parameter on the Ip and Ep of AFB2 was studied. In each experiment, one of the parameters was varied while others were kept constant. 2.2.3.2a

Effect of Scan Rate (υ)

After the best condition of pH was chosen, the effect of υ was carried out. The υ were varied from 20 to 100 mV/s while maintaining other parameters constant. Subsequently a purge of nitrogen gas for 120 s was employed between cathodic stripping cycles. A graph of Ip against υ was then plotted and the best υ was then selected.

80 2.2.3.2b

Effect of Accumulation Potential (E acc)

The general procedure was followed with the best condition of pH and υ set to the required value. Effect of Eacc was carried out with other parameters kept unchanged as previously mentioned. A graph of Ip against Eacc was then plotted. 2.2.3.2c

Effect of Accumulation Time (tacc)

Using the best condition for Eacc, tacc and pH of BRB solution, general procedure was followed where a series of scans with increasing tacc were carried out. A graph of Ip against tacc was then plotted. 2.2.3.2d

Effect of Initial Potential (Ei)

The effect of Ei to the Ip using the chosen previous optimised parameters was carried out where a series of scans with different Ei while Ef kept constant were performed. The effect of Ei was carried out with variation of 0 V to -1.10 V and increasing towards more negative potential until it reached the required maximum Ip. A graph of Ip against Ei was then plotted. 2.2.3.2e

Effect of Pulse Amplitude

The best conditions of other parameter were chosen and the pulse amplitude was optimised as the final step in this optimisation procedure. The effect of pulse amplitude was carried out with variation of 30 to 100 mV. A graph of Ip against pulse amplitude was then plotted. 2.2.3.3

Method Validation

Validation of the method was examined via evaluation of linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, repeatability, reproducibility,

81 recovery, robustness and ruggedness. Multiple scanning of blank before addition of AFB2 standard solution was performed. LOD was calculated by standard addition of low concentration of aflatoxins until a response was obtained that is significantly different from the blank sample (Barek et al., 2001a). The intra-assay and inter-assay precision for AFB2 was calculated from repeated analysis of certain concentration of AFB2 during one working day and on different days respectively. Recovery study for the accuracy of the method were performed by adding an amount of AFB2 into the eluate of real samples and after extraction and clean-up procedures before being analysed for AFB2 and percentage recovery of AFB2 was calculated. The robustness, as a measure of procedure’s capability to remain unaffected by small variations of some important procedure conditions, was examined by analysis of 0.1 µM AFB2 (31.4 ppb) under the successive variation of pH of the supporting electrolyte (pH 8.5 – 9.5), Eacc (from - 0.59 to - 0.61 V) and tacc (75 – 85 s). The ruggedness of the measurement was examined by applying the proposed procedure to assay of aflatoxin using two different models of voltammetry instruments that were BAS and Metrohm voltammetry analysers. For other aflatoxins (AFB1, AFG1 and AFG2) the same procedures were followed as mentioned for AFB2 including method validation as well. For all cases, each calibration curve was constructed and the same statistical parameters were evaluated. 2.2.3.4

Interference Studies

2.2.3.4a

Effect of Cu (II), Ni (II), Al (III), Pb (II) and Zn (II)

The effect of all the above metals on the measurement of 0.1 µM of aflatoxin in BRB pH 9 was carried out by adding a series of concentration of all metal standard solutions into the aflatoxin solution in the voltammetric cell started from 0.1 to 1.0 µM concentration. The graphs of Ip of respective aflatoxin against the concentration of all standard solutions were plotted.

82 2.2.3.4b

Effect of Ascorbic Acid, β-CD and L-cysteine

The effect of ascorbic acid, β-CD and L-cysteine on the measurement of 0.1µM of aflatoxin in BRB pH 9 were carried by adding a series of concentration of ascorbic acid, β-CD and L-cysteine solutions into the aflatoxin solution in the voltammetric cell from 0.1 to 1.0 µM concentration of each compound. The graphs of Ip of respective aflatoxin against the concentration of added acorbic acid, β-CD and L-cysteine were plotted. 2.2.3.5

Modified Mercury Electrode with Poly-L-Lysine (PLL)

10 ml of BRB solution at pH 9.0 was pipetted into voltammetric cell followed with 10 µl of PLL and 0.1 µM aflatoxins. The solution was purged for 10 minutes before general procedure for voltammetric determination was applied. Ip of 0.1µM AFB2 was measured. The procedure was repeated by using different pH of BRB. 2.2.4

Square Wave Cathodic Stripping Voltammetry (SWSV)

All optimised parameters which were obtained using DPCSV were applied using SWSV for determination of 0.1 µM of each aflatoxin. 2.2.4.1 SWSV Parameters Optimisation

All SWSV parameters such as pulse amplitude, voltage step and frequency were optimised to obtain maximum Ip and defined peak for AFB2. For this optimisation procedure, 0.1 µM AFB2 was used. 2.2.4.2 SWSV Determination of All Aflatoxins

Other aflatoxins with concentration of 0.1 µM have been measured using SWSV technique with optimised parameters. The results of Ip and Ep of all aflatoxins were

83 compared with those obtained by DPCSV technique. The effect of concentration on Ip and Ep of aflatoxins has been observed. The plot of Ip versus concentration of respective aflatoxin was constructed and all statistical parameters were evaluated. 2.2.5 Stability Studies of Aflatoxins

These studies were carried out by a series of experiments according to the procedures elaborated below; 2.2.5.1 Stability of 10 ppm Aflatoxins

Stability of 10 ppm aflatoxin stock solutions prepared in benzene: acetonitrile (98%) was monitored using UV-VIS spectrophotometer using 98% of benzene: acetonitrile as a blank. 1 ml of sample was scanned three times starting from λ of 500 nm to 280 nm. This measurement was done every month from first day of preparation for 12 months period time. The concentration of each aflatoxin were calculated using the formula as stated in Appendix D. 2.2.5.2 Stability of 1 ppm Aflatoxins

Stability of 1ppm aflatoxin standard solutions prepared in BRB pH 9.0 was performed using DPCSV method. In this experiment, 0.1 µM of each aflatoxin was scanned every month from first to 6th month. The graph of Ip current against months of keeping period was plotted. 2.2.5.3 Stability of 0.1 µM Aflatoxins Exposed to Ambient Temperature

Stability study of 0.1 µM aflatoxins in BRB pH 9.0 which was exposed to the ambient temperature in voltammetric cell was performed using DPCSV method. In this experiment, 0.1 µM of each aflatoxin was scanned every hour from 0 to 8th hour

84 exposure time. The graph of Ip against exposure time was plotted. Nitrogen gas was purged for 10 min before performing each scan. 2.2.5.4 Stability of 0.1 µM Aflatoxins in Different pH of BRB

Stability of 0.1µM aflatoxins in different pH of BRB (6 to 11.0) were observed. For each pH value, the Ip of aflatoxin was measured for 3 hours. The graph of Ip against exposure time for each pH of BRB was plotted. 2.2.6

Application to Food Sample

Fifteen batches of real samples (groundnut) were purchased and labeled with S01 to S15 were used in this study. The samples were subjected to the extraction and clean-up procedure prior voltammetric analysis using DPCSV and SWSV techniques. A few techniques which were named as Technique 1, 2 and 3 were studied for extraction and clean-up of real samples. 2.2.6.1 Technique 1

50 g of groundnut samples were weighed into 250 ml blender, then 100 ml of acetonitrile-water (9:1) solution were added and shaken for 1 hour. The extraction solution was filtered through a pre-pleated filter paper to remove solid material and extract was collected and kept for analysis (Pena et al., 2002). 2.2.6.2 Technique 2

Groundnut samples were first ground in a household blender at high speed for 3 minutes. For extraction, 10 ml of methanol-water (80:20) was added to 5g of sample, followed by 5 ml hexane. The suspension was hand shaken for 3 minutes and then passed through Whatman No. 4 filter paper. The aqueous layer was diluted 1 in 10 for the assays to avoid any interference from methanol, hence the consequence aflatoxin’s

85 concentration in the extracted solution was 1/20 th that in groundnut samples. The extraction took approximately 15 minutes to perform (Garden et al., 2001) 2.2.6.3 Technique 3

This technique is adapted by The Chemistry Department, Penang Branch, Ministry of Science, Technology and Innovation (MOSTI), Pulau Pinang (2003) as shown in Appendix E. 1.0 ml of final solution in chloroform was taken and put into an amber bottle followed with degassing and re-dissolved in 1.0 ml BRB at pH 9.0 before spiking into voltammetric cell for analysis. 2.2.6.4

Blank Measurement

For blank measurement, the procedure of extraction and clean-up steps were followed as previously mentioned in Appendix E without addition of real sample. The blank consisted of methanol, 0.1 N HCl, 15% ZnSO4 and chloroform. 1.0 ml of final solution was collected from extraction procedure in chloroform, degassed and redissolved in 1.0 ml of BRB at pH 9.0. 2.2.6.5

Recovery Studies

Recovery studies for aflatoxins in groundnut samples were performed using DPCSV and SWSV techniques. For this study, a certain volume of 1.0 ppm of aflatoxins as listed in Table 3.3 were injected into 20.0 ml of eluate from blended real sample (Ammida et al., 2004). The sample with injected aflatoxins was then mixed with 20.0 ml zinc sulphate, 15%. The total amount of added aflatoxins in the cell obtained by respective injected volume is shown in Table 2.1. The solution was well mixed for 30 s and subjected to extraction procedure as previously mentioned. Sample solution was prepared by degassed of 1 ml of chloroform until dryness followed with dissolution in BRB at pH 9.0.

86 Injected volume of aflatoxins into eluate of groundnut and the final

Table 2.1

concentrations obtained in voltammetric cell Injected volume of 1 ppm aflatoxins (ml)

2.2.6.6

Final concentration in voltammetric cell (ppb)

1.6

3.0

5.1

9.0

9.3

15.0

Voltammetric Analysis

Voltammetric analysis of blank, recovery and real samples were carried out using DPCSV and SWSV techniques. For each sample, 200 µl of the solution was spiked into 10 ml supporting electrolyte followed with general voltammetric determination procedure. Voltammograms of each samples were recorded and any peak appeared was observed. For determination of aflatoxin in real sample, standard addition method was applied by spiking 10 ppb of aflatoxin standard followed with general procedure for voltammetric analysis. The amount of aflatoxin in groundnut sample was calculated according to the formula as stated below; Aflatoxin (ppb) = P’ / P x C x 12.5

(2.0)

where; P’

=

Ip of sample (nA)

P

=

Ip of aflatoxin standard (after substract from P’) (nA)

C

=

Concentration of aflatoxin spiked in the cell (ppb)

12.5

=

Factor value after the sample weight, volume of chloroform used in the extraction and preparation of injection sample have been considered

87 The detailed information on how this formula was formulated are shown in Appendix F The results were compared with that obtained by HPLC technique. The HPLC system used was a Waters model comprising WATERS 600 controller, WATERS 717 auto sampler, WATERS temperature control module and MILLENIUM chromatography manager, equipped with a model WATERS 420-AC fluorescence detector filled with standard optical filters with 338 nm bandpass excitation filter and 425 nm longpass emission filter, gain; 16 and maximum span. For chromatographic separation, a Nova-Pak C-18 steel column (150 x 3.9 mm, 4um particle size) was employed. The separation was carried out at room temperature using, as the mobile phase of 70% water, 20 % methanol and 10 % acetonitrile with a flow rate of 1.6 ml / min at column temperature of 30° C. The injection volume was 30 µl with 12 min for running time.

88

CHAPTER III

RESULTS AND DISCUSSION

3.1

Cyclic Voltammetric Studies of Aflatoxins

Cyclic voltammetric (CV) experiment is normally carried out for the initial electroanalytical studies of any compound to obtain information on electroanalytical properties of the compounds before being investigated in depth using other electroanalytical techniques is carried out (Wang, 2000). In this work, all aflatoxins (AFB1, AFB2, AFG1 and AFG2) have been initially analysed by this technique. The main objective of this experiment was to obtain the electroanalytical properties of aflatoxin at different scan rates in various pH of BRB solution. Any current peak that appeared from the voltammogram was evaluated to confirm the type of reaction of aflatoxin on the mercury electrode. The repetitive cyclic voltammetry were also performed at certain CV parameters such as scan rate of 200 mV/s in BRB solution with pH of 9.0. This experiment was carried out to observe the process of accumulation of aflatoxins on mercury electrode with longer accumulation time. Various pH medium of BRB have been used in these experiments to observe any involvement of hydronium ion in the electrode processs of aflatoxins on the mercury electrode (Kamal et al., 1996). The peak potentials of these compounds which were measured throughout this work were against an Ag/AgCl as the reference electrode.

89 3.1.1

Cathodic and Anodic Cyclic Voltammetric of Aflatoxins

The parameters set up for cathodic cyclic voltammetric measurement were initial potential (Ei ) = 0 mV, high potential (EH ) = 0 mV, low potential (EL) = -1500 mV, scan rate (ν) = 200 mV/s and sensititvity = 1 µA/V. A BRB solution with pH 9.0 was initially used as the supporting electrolyte following the previous experiment carried out by Smyth et al. (1979) using DPP technique. The result that were obtained showed that the BRB solution with pH 9.0 was the best medium for reduction of aflatoxins on the mercury electrode. CV experiments results also revealed that the BRB pH 9.0 is the optimum pH for this reduction to take place. Figure 3.0 shows the Ip of 0.6 µM AFB1 obtained by cathodic cyclic voltammetry in different pH of BRB. The Ep of AFB1 was shifted to the more negative direction with increasing pH as shown in Figure 3.1 indicating that proton was involved in the reduction of AFB1 (Sun et al., 2005).

30

Ip (nA)

25 20 15 10 5 0 5

6

7

8

9

10

11

12

13

pH of BRB

Figure 3.0

Cathodic peak current of 0.6 µM AFB1 in various pH of BRB obtained

by cathodic cyclic voltammetry. Ei = 0, Elow = -1.5 V, Ehigh = 0 and scan rate = 200 mV/s.

90 From the Figure 3.0, the curve rises from pH 6.0 to 9.0 then goes down from pH 9.0 to 12.0. According to Smyth et al. (1979), for pH 6.0 to 8.0, the reduction peak of AFB1 was caused by the reduction of double bond in benzene ring which conjugates to the keto group and followed by the protonation of molecule that preceded the reduction until it achieved maximum reduction at pH 9.0. The possible mechanism for this reaction is shown in Figure 3.2.

1.34

Ep (-m V)

1.33 1.32 1.31 1.3 1.29 1.28 1.27 5

6

7

8

9

10

11

12

13

pH of BRB

Shifting of Ep of AFB1 with incresing pH of BRB. Parameter conditions

Figure 3.1

are the same as in Figure 3.0.

O

O

O

O

H

H

O

O

2e2H+

OCH3

Figure 3.2

OCH3

Mechanism of reduction of AFB1 in BRB at pH 6.0 to 8.0

91

For pH 10 to 12.0, due to decreasing number of hydrogen ions in the supporting electrolyte that is involved in the reduction process, anion radical was dimerised and produced a reduction peak. Their mechanism is shown in Figure 3.3.

O

O

O

O

O

O

2e -

2

2 O CH 3

OC H3

2 H 2O

D im er

Figure 3.3

+ 2 OH-

Mechanism of reduction of AFB1 in BRB at pH 9.0 to 11.0

Using BRB at pH 9.0 as the optimum medium for the supporting electrolyte, cathodic cyclic voltammograms of 1.3 µM of all aflatoxins were obtained where all aflatoxins produced a sharp and well defined cathodic wave around -1.315 V for both AFB1 and AFB2, -1.245 V for AFG1 and -1.250 V for AFG2 with a peak current of 42 nA, 30 nA, 47 nA and 39 nA for AFB1, AFB2, AFG1 and AFG2 respectively. The peak was located not far from the peak of hydrogen over potential which was at -1.450 V. For all aflatoxins, no oxidation wave appeared in the anodic branch, which indicates that the aflatoxins reductions are irreversible. Cyclic voltammograms of all aflatoxins are shown in Figures 3.4 to 3.7. The effect of initial potential (Ei)) on cathodic peak of AFB1 was investigated. Varying Ei caused no extra peak to appear for both cathodic and anodic waves which

92 show that the Ei did not produce any significant change on the cathodic peak of AFB1. The Ip were almost the same for Ei at 0 to -0.8 V which was around 22 nA.

Figure 3.4

Cathodic cyclic voltammogram for 1.3 µM AFB1, obtained at

scan rate of 200 mV/s, Ei = 0, Elow = -1.5 V and Ehigh = 0 in BRB solution at pH 9.0.

Figure 3.5

Cathodic cyclic voltammogram for 1.3 µM AFB2 in BRB solution at pH

9.0. All parameter conditions are the same as in Figure 3.4.

93

Figure 3.6

Cathodic cyclic voltammogram for 1.3 µM AFG1 in BRB solution at pH

9.0. All parameter conditions are the same as in Figure 3.4.

Figure 3.7

Cathodic cyclic voltammogram for 1.3 µM AFG2 in BRB solution at pH

9.0. All parameter conditions are the same as in Figure 3.4. For Ei of more than -0.80 V, the Ip decreased to 17 nA as shown in Figure 3.8. This is because the presence of Zn+2 in BRB solution facilitates the migration and adsorption of aflatoxin towards the working electrode. This is proven by the study of

94 Zn +2 using CV technique with various Ei and the results are shown in Figure 3.9. There is no significant change on Ep of AFB1 (-1.30 V) with changing Ei . 30

I p (nA)

25 20 15 10 5 0 0

0.2

0.4

0.6

0.8

1

1.2

1.4

Ei (-V)

Figure 3.8

Effect of Ei to the Ip of 0.6 µM AFB1 in BRB at pH 9.0 obtained by

cathodic cyclic voltammetry. Parameter conditions are the same as in Figure 4.4.

Ip (nA)

20 15 10 5 0 0

0.2

0.4

0.6

0.8

1

1.2

Ei (-V) Figure 3.9

Effect of Ei to the Ip of 0.1 µM Zn2+ in BRB at pH 9.0 obtained by

cathodic cyclic voltammetry. Parameter conditions are the same as in Figure 3.4. Anodic cyclic voltammetry measurement was carried out using the same parameters with the exception of Ei = -1500 mV (versus Ag/AgCl). The anodic cyclic voltammograms confirmed that the reduction reaction of all aflatoxins are irreversible

95 whereby only cathodic peak appeared but with a slightly higher current of 46.76 nA at 1.318 V for AFB1, 35 nA at -1.311 V for AFB2, 52 nA at 1.243 V for AFG1 and 50 nA at -1.259 V for AFG2. This may be due to longer time of anodic scanning itself, which gave a longer time for accumulation of aflatoxins on the mercury electrode surface compared to the cathodic measurement. For example, anodic cyclic voltammogram of AFB2 is shown in Figure 3.10.

Figure 3.10

Anodic cyclic voltammogram of 1.3 µM AFB2 obtained at scan rate of

200 mV/s, Ei = -1.5 V, Elow = -1.5 V and Ehigh = 0 in BRB solution at pH 9.0. Standard additions of each aflatoxin shows that the peak current at their respective potential increases with increasing concentrations. The voltammograms of AFB2 (Figure 3.11) shows that the peak current at -1.315 V increases with increasing AFB2 concentration. This dependence is represented in Figure 3.12. The corresponding equation for this dependence is given below; Ip (nA) = 21.65 x (µM) + 6.605

(R2 = 0.9857, n = 4)

(3.0)

No extra peak was observed with this standard addition either at the anodic direction or cathodic direction. This result reveals that the appeared peak was due to the respective aflatoxin and does not from other electroactive compounds.

96

Figure 3.11

Effect of increasing AFB2 concentration on the peak height of cathodic

cyclic voltammetric curve in BRB at pH 9.0; (a) 1.30 µM, (b) 2.0 µ M (c) 2.70 µM and (d) 3.40 µM. Parameter conditions are the same as in Figure 3.4

100

Ip (nA)

80 60 40

y = 21.65x + 6.605

20

R2 = 0.9857

0 1

1.5

2

2.5

3

3.5

4

[AFB2] / uM Figure 3.12

Peak height of reduction peak of AFB2 with increasing concentration of

AFB2. Experimental conditions are the same as in Figure 3.4.

97 For other aflatoxins, the dependence of current peak to their concentrations in BRB at pH 9.0 is listed in Table 3.0. The regression equations of this relationship show that the peak heights of aflatoxins are correlate well with the concentration of aflatoxins. Cathodic cyclic voltammograms of other aflatoxins for increasing concentrations are shown in Appendix G. The plots of Ip versus concentrations for other aflatoxins are shown in Appendix H. Table 3.0

The dependence of current peaks of aflatoxins to their concentrations

obtained by cathodic cyclic measurement in BRB at pH 9.0

Aflatoxin

Regression equation for increasing

R2

concentration from 1.30 to 3.40 µM (n=4)

AFB1

y = 22.451x + 10.989

0.9899

AFB2

y = 21.650x + 6.605

0.9807

AFG1

y = 24.756x + 13.072

0.9812

AFG2

y = 33.55x + 1.435

0.9914

Repetitive cathodic stripping voltammetry studies for all aflatoxins were also carried out in order to observe whether any adsorption phenomenon took place at the surface of mercury electrode as reported by Ghoneim et al. (2003a). The results show that the reduction peak of all aflatoxins increases with the number of cycles, which may be attributed to adsorption of aflatoxins at the mercury electrode surface (Farias et al., 2003). No other cathodic peak was observed for all cases which show that no other species was produced by the reduction of aflatoxins. The peak potentials of all

98 aflatoxins were shifted to less negative direction with increasing number of cycles. For example, repetitive cathodic cyclic voltammograms of AFB2 is shown in Figure 3.13. Figures 3.14 and 3.15 show the increasing of peak current of AFB2 cathodic peak and shifting of its peak potential to less negative direction with increasing number of cycle respectively. For other aflatoxins, their repetitive cyclic voltammogram are shown in Appendix I.

Figure 3.13

Repetitive cathodic cyclic voltammograms of 1.3 µM AFB2 in BRB

solution at pH 9.0. Experimental conditions are the same as in Figure 3.4. 60

Ip (nA)

50 40 30 20 10 0 1

2

3

4

5

No of cycle

Figure 3.14

Increasing Ip of 1.3 µM AFB2 cathodic peaks obtained from repetitive

cyclic voltammetry. Experimental conditions are the same as in Figure 3.4.

99

1316

Ep (-mV)

1312

1308 1304

1300 0

1

2

3

4

5

6

No of cycle

Figure 3.15

Peak potential of 1.3 µM AFB2 with increasing number of cycle

obtained by repetitive cyclic voltammetry.

The adsorption of aflatoxins on the electrode surface is expected to be due to the aflatoxins chemical structures that consist of many functional groups such as ketones, ester and ether. The presence of these functional groups increases the polarity and adsorption (Volke and Liska, 1994) of the compound and may be attributed to the adsorption process taking place on the electrode surface. Furthermore, due to the presence of the phenyl ring, aflatoxins have surface activity and are readily adsorbed onto mercury surface (El-Hefnawey et al., 2004). However, a further increase in the repeatitive cycle tends to slow down the increment of the Ip due to the formation of a double layer at the mercury electrode (Fifield and Kealey, 2000).

The effect of increasing scan rate, υ (from 20 to 500 mV/s) to the Ep and Ip of aflatoxins cathodic peaks were observed under the same experimental conditions. Linear relationship was observed between the log Ip versus log υ for all aflatoxins with slope values of 0.5346, 0.5097, 0.5623 and 0.5439 for AFB1, AFB2, AFG1 and AFG2 respectively. The slope value of more than 0.5 indicates that the diffusion current

100 produced by the reduction of all aflatoxins on the mercury electrode are influenced by adsorption in the electrochemical process at the electrode surface (Gosser, 1994 and Yaridimer and Ozaltin, 2001). For example, a plot of log Ip versus log υ for AFB2 is shown in Figure 3.16.

1.9 1.7 Lo g Ip

1.5 1.3 1.1 0.9

y = 0.5097x + 0.4115

0.7

R2 = 0.995

0.5 1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

log scan rate Figure 3.16

Plot of log Ip versus log υ for 1.3 µM AFB2 in BRB solution at pH 9.0.

Experimental conditions are the same as in Figure 3.4. Plotting Ep versus log υ for scan rates in the range of 20 to 500 mV/s for all aflatoxins, linear graphs were obtained according to the equation: a) Ep (-mV) = 61.415 log υ + 1171.6 (R2 = 0.9987, n = 9) for AFB1

(3.1)

b) Ep (-mV) = 40.065 log υ + 1223.2

(R2 = 0.9936, n = 9) for AFB2

(3.2)

c) Ep (-mV) = 48.484 log υ + 1134.8

(R2 = 0.9978, n = 9) for AFG1

(3.3)

d) Ep (-mV) = 49.297 log υ + 1146.0

(R2 = 0.9984), n= 9) for AFG2

(3.4)

The Ep linearly shifted to the more negative direction which confirms the irreversibility of the reduction processes on the mercury electrode (de Betono et al, 1996 and Arranz et al. 1999). Figure 3.17 shows a plot of Ep of AFB2 versus log υ in the range of 20 to 500 mV/s. For other aflatoxins, these plots are shown in Appendix J.

101

1340

Ep (-mV)

1330 1320 1310 1300 1290

y = 40.065x + 1223.3

1280

R2 = 0.9936

1270 1.2 1.4 1.6 1.8

2

2.2 2.4 2.6 2.8

log scan rate Figure 3.17

Plot of Ep versus log scan rate for 1.3 µM AFB2 in BRB solution at pH

9.0 A linear plot was obtained for the effect of scan rate on Ip of aflatoxins with 2 linear regions. Taking AFB2 for an example, in the first region which is for the faster scan rate (100 to 500 mV/s) the equation was linear according to the equation Ip = 0.0759x + 24.566 (R2 = 0.9917, n = 5) as shown in Figure 3.18. At the second region where the scan rate was slow (20 to 80 mV/s), the linear equation is Ip = 0.2639x + 6.16 (R = 0.9979, n = 5) which was also linear. Due to the slope of slow scan rate region that is higher than the fast scan rate region, it can be concluded that the reduction of aflatoxins on the mercury electrode prefers a slow reaction. The plots for other aflatoxins are illustrated in Appendix K. As far as the chemical structures of AFB1, AFB2, AFG1 and AFG2 are concerned, the main difference of AFB1 and AFG1 with AFB2 and AFG2 is the absence of double bond in the terminal furan ring of AFB2 and AFG2 compared to AFB1 and AFG1. From the result, it suggests that this double bond does not play a major role in the reduction process at the mercury electrode since all of them gave single cathodic peak at very close potentials. However, the double bond in the benzene

102 ring which conjugates with the keto group has the potential to be reduced on the mercury electrode and produce cathodic peak.

70 60

Ip (nA)

50 40 30 20 10 0 0

50 100 150 200 250 300 350 400 450 500 550 Scan rate (mv/s)

Figure 3.18

Plot of Ip versus υ for 1.3 µM AFB2 in BRB solution at pH 9.0.

Experimental conditions are the same as in Figure 3.4. From overall results of CV studies, all aflatoxins reduced at mercury electrode gave a single cathodic peak with Ip at about 40 ± 5.0 nA for the concentration of 1.3 µM with Ep between -1.250 V and -1.315 V. All the aflatoxins were adsorbed onto the mercury electrode. Due to these properties, an adsorptive cathodic stripping voltammetric technique was developed to obtain more sensitive method for the determination of trace levels of aflatoxins. 3.2

Differential Pulse Cathodic Stripping Voltammetry (DPCSV) of AFB2

In this experiment, AFB2 was used to represent the aflatoxins since all of them have the same electrochemical behaviour as those found in previous experiments. Since the results from CV studies showed that all aflatoxins underwent reduction

103 process only, AFB2 was analysed by DPCSV technique using these parameters; Ei = 0, Ef = –1.50 V, Eacc = 0 mV, no tacc at υ of 50 mV/s. In this study, AFB2 standard solution was redissolved in BRB at pH 9.0 after the organic solvent had been completely removed to avoid any effect on the diffusion rate of electroactive species and the electrical double layer thickness which may increase the current resistance and further increase the noise level (Kotoucek et al. 1997). BRB solution at pH 9.0 was used as the supporting electrolyte because from the previous experiment by CV technique, the BRB solution at pH 9.0 was the best solution for the determination of aflatoxin. The initial result in Figure 3.19 shows that a single peak with a peak height (Ip) about 5.0 nA was observed at a peak potential (Ep) of -1.256 V for 1.0 uM AFB2. The other peak which appears at Ep of -0.92 V is the Zn peak from the contamination of BRB solution. The single peak at -1.256 V was due to the AFB2 reduction peak since no extra peak was observed as previously confirmed by the CV technique.

Figure 3.19

DPCS voltammograms of 1.0 µM AFB2 (Peak I) in BRB at pH 9.0 (a) at

tacc = 0 (b) and 30 s (c). Other parameter conditions; Ei = 0, Ef = -1.50 V, Eacc = 0 V, υ = 50 mV/s and pulse amplitude = 100 mV. Peak II is the Zn peak.

104 The effect of tacc on the Ip of AFB2 peak was also examined. When tacc was set to 30 s, the Ip of AFB2 increased to 22 nA and at the same time, no increase for the Ip of the Zn peak (Figure 3.19). This result suggests that AFB2 has been actively adsorbed on the mercury electrode surface. This phenomenon is similar to the previous result found by the CV study. 3.2.1

Optimisation of Conditions for the Stripping Analysis

3.2.1.1

Effect of pH and Type of Supporting Electrolyte

The adsorptive cathodic stripping voltammetric response for 2.0 µM AFB2 was examined in BRB solution over the pH range from 3.0 to 13.0 using cathodic stripping voltammetry, under the operational conditions: Ei = 0 V, Ef = -1.5 V, Eacc = 0 V, tacc = 30 s, υ = 50 mV/s and pulse amplitude = 100 mV. The results are presented in Figure 3.20.

Figure 3.20

DPCS voltammograms for 2.0 µM AFB2 in BRB (Peak I) at different

pH values: (a) 6.0, (b) 7.0 (c) 8.0 (d) 9.0 (e) 11.0 and (f) 13.0. Eacc = 0 V, tacc = 30 s, υ= 50 mV/s and pulse amplitude = 100 mV. Peak II is the Zn peak.

105 Since only a single cathodic peak for AFB2 was obtained, it can be concluded that the AFB2 molecule exhibits one reduction peak over the studied pH. It was found that for pH less than 4.0, no peak was observed. At pH 4.0 and less, the peak of AFB2 was probably overlapped by hydrogen over potential which appeared at less negative potential (Abu Zuhri et al, 2000). In acidic conditions, the concentration of hydrogen ion is higher and more readily reduced at the mercury electrode producing a peak at potential value around -1.20 V. The single and characteristic AFB2 peak was initially observed at pH 5.0 with Ip of 11.0 nA and Ep located at -1.119 V. The increase in the pH gradually increased the Ip until it reached its maximum value at pH 9.0 (45 nA at Ep of -1.192 V). When there is further increase of pH beyond pH 9.0, the Ip decreased until it disappeared at pH 13.0 as shown in Figure 3.21. This result is not contradictory with that obtained by Smyth et al. (1979) when they studied the effect of pH of BRB on the peak height of AFB1 using the differential pulse polarographic (DPP) technique. They also found that BRB at pH 9.0 gave the highest Ip of AFB1.

Ip (nA)

.

60 50 40 30 20 10 0 4

6

8

10

12

pH Figure 3.21

Dependence of the Ip for AFB2 on the pH of 0.04 M BRB solution.

AFB2 concentration: 2.0 µM, Ei = 0, Ef = -1.5V, Eacc = 0, tacc = 30 sec, υ = 50 mV/s and pulse amplitude = 100 mV.

106 The sharp decrease in Ip at pH 10.0 to 12.0 and no current peak obtained for pH 13.0 indicates that there is deficiency in the amount of hydrogen for reduction process to take place, hence the peak height decreased. At pH 13.0 no peak was observed which indicates that at this pH, the chemical structure of AFB2 may be totally damaged or another reaction has taken place under alkaline medium. This finding was confirmed by measuring AFB2 in BRB at pH 9.0 and followed with the lower pH such as 4.0 and subsequently increased to pH 9.0. The measurements were then carried out in the higher pH such as 10.0, 11.0, 12.0 and 13.0 and then back to 9.0 without changing the

70

70

60

60

50

50

40

40

I p (n A )

I p (n A )

solution. The result of this experiment is shown in Figure 3.22.

30

30

20

20

10

10

0

0 9

6

4 pH of BRB

(a) Figure 3.22

6

9

9

10

11

12

13

12

10

9

pH of BRB

(b)

Ip of 2.0 µM AFB2 obtained in BRB (a) pH from 9.0 decreases to 4.0

and reincrease to 9.0 (b) pH from 9.0 increase to 13.0 and redecrease to 9.0. Results show that at pH 4.0, no peak was observed but while the pH was increased to 6.0 and pH 9.0, the peak appeared which indicated that the structure of AFB2 was not damaged by the strong acidic condition. When pH was adjusted to pH 13.0, the peak dissapeared and no peak was observed even when the pH was re-adjusted to 9.0 which showed that at pH 13.0, the structure of AFB2 was totally damaged or transformed to nonelectroactive compound in the strong basic media.

107 The above finding was further confirmed by measuring the absorbance of the AFB2 using UV-VIS spectrophotometer. The results from UV-VIS technique are shown in Figures 3.23a, 3.23b and 3.23c for AFB2 in BRb pH 6.0, 9.0 and 13.0 respectively. The results show that no absorbance was obtained at wavelength (λ) of 365 nm for AFB2 in BRB at pH 13.0 while for AFB2 in BRB at pH 6.0 and 9.0 the absorbance were 0.104 and 0.102 respectively. This was due to the fact that in BRB pH 13.0 the functional group (ketone), one of the UV chromophore which conjugates to double bond in benzene ring is damaged due to the opening of this ring by the high basic condition as reported by Janssen et al. (1997).

(b)

(a)

(c) Figure 3.23

13.0.

UV-VIS spectrums of 1 ppm AFB2 in BRB at pH (a) 6.0, (b) 9.0 and (c)

108 Figure 3.24 shows the opening of the lactone ring when AFB2 reacts with high alkaline solution. When no keto group conjugated with the double bond, it is difficult for the carbon-carbon double bond in the benzene to be reduced (Smyth and Smyth, 1978) which produce no reduction peak.

O

O

O

HO

O

O

OCH3

O

Figure 3.24

O O

OCH3

Opening of lactone ring by strong alkali caused no peak to be observed

for AFB2 in BRB at pH 13.0. This results shows that the reduction of AFB2 is totally dependent on the supporting electrolyte which suggests that the reduction of AFB2 involves the reaction of AFB2 with hydrogen ions originating from the supporting electrolyte (Abu Zuhri, 2000, Herdenandez et al., 1977 and Rodriguez et al. 2005). The peak response was also examined in the presence of different buffers such as phosphate and acetate buffers at the pH 9.0. BRB was selected as the most suitable supporting electrolyte since it gave the highest Ip and good repeteability, as shown in Table 3.1. It was found that the presence of peak of AFB2 reduction is strongly influenced by pH and the buffer constituent. The supporting electrolyte has two roles to play. The concentration of the supporting electrolyte regulates the electrical resistance of the cell and it also controls the migration of ions between the working and secondary electrode (Riley and Tomlinson, 1987).

109 Table 3.1

Effect of buffer constituents on the Ip of 2.0 µM AFB2 at pH 9.0.

Experimental conditions are the same as in Figure 3.21

Buffer solution

Ip (nA)

Ep (V)

% RSD

(n =5)

Britton Robinson

50.08

-1.192

0.51

Carbonate

27.82

-1.130

1.19

Phosphate

43.74

-1.140

1.35

The effect of different concentrations of BRB at pH 9.0 as the supporting electrolyte has been carried out and the results are shown in Figure 3.25 which shows that BRB with 0.04 M concentration gave the highest Ip of AFB2 compared with other studied concentrations. In 0.08 M, the Ip decreases due to the excess of supporting electrolyte concentration causing the ionic migration of the aflatoxin to be reduced (Riley and Tomlinson, 1987).

60

Ip (nA)

50 40 30 20 10 0 0.02 M

0.04 M

0.08 M

[BRB]

Figure 3.25

Ip of 2.0 µM AFB2 in different concentration of BRB at pH 9.0.

Experimental conditions are the same as in Figure 3.20.

110 The effect of different pH and concentrations of BRB has been studied and the results are shown in Figure 3.26. The figure shows that BRB with concentration of 0.04 M at pH 9.0 is the most suitable solution for the voltammetric determination of AFB2. This can be explained that for 0.04 M at pH 9.0, the migration and adsorption of AFB2 at the working electrode was maximum as compared to other conditions. Voltammograms of AFB2 in different concentrations of BRB at pH 9.0 are shown in Figure 3.27 60

Ip (nA)

50 40

pH = 6.0

30

pH = 7.0

20

pH = 9.0

10 0 0.02 M

0.04 M

0.08 M

[BRB]

Figure 3.26 Ip of 2.0 µM AFB2 in different pH and concentrations of BRB

Figure 3.27

DPCS voltammograms of 2.0 µM AFB2 (peak I) in (a) 0.04 M, (b) 0.08

M and (c) 0.02 M BRB at pH 9.0 as the blank. Ef = -1.5 V, Eacc = 0 v, tacc = 30 s, υ = 50 mV/s and pulse amplitude = 100 mV. Peak II is the Zn peak

111 As determined in previous CV and DPCSV experiments, AFB2 were reduced at the mercury electrode. Based on the chemical structure of aflatoxin, there are a few sites that may undergo this process. There are double bonds in the terminal furan rings for AFB1 and AFG1 and a double bond in the benzene ring which is conjugated with the ketone group for all aflatoxins. Further investigations have been carried out to confirm which site is actually involved in this reduction reaction at the mercury electrode by voltammetric study of separate basic compounds that form aflatoxins which are 2,3 dihydrofuran and coumarin. Another compound is tetrahydrofuran but it does not have any double bond in their chemical structure which may not be reducable at the electrode, hence no detailed investigation has been carried out. Their chemical structures are shown in Figures 3.28a to 3.28c. Other possibility of the chemical structure that form aflatoxin compound was not studied due to the unavailability of the compound.

Figure 3.28

O

(a)

(b)

O

O

O

(c)

Chemical structures of (a) 2,3 dihydrofuran (b) tetrahydrofuran and (c)

coumarin Voltammograms of 0.02 to 0.2 µM of 2,3 dihyrofuran in BRB pH 9.0 are shown in Figure 3.29. There is a small peak observed at -1.21 V which has not significantly increased when the concentration was increased. The result indicates that 2,3 dihydrofuran is not reduced at the mercury electrode which indicates that the reduction of AFB1 and AFG1 is not due to the reduction of double bond in their terminal furan rings.

112

Figure 3.29

Voltammograms of 2,3 dihydrofuran (peak I) for concentrations from (b)

0.02 to 0.2 µM in (a) BRB at pH 9.0. Peak II; Zn peak. The second compound that may undergo reduction at the mercury electrode is coumarin (Figure 3.27c). Increasing the concentration of coumarin yields a linear relationship of Ip corresponding to an equation of y = 25.877x – 35.5 (R2 = 0.9953). A plot of Ip versus concentration of coumarin is shown in Figure 3.30.

500

Ip (nA)

400 300 200

y = 25.877x - 35.5

100

R2 = 0.9953

0 0

5

10

15

20

[Coumarin] x 10-5M

Figure 3.30

Dependence of Ip of coumarin to its concentrations

113 The voltammograms of coumarin with increasing concentration are represented in Figure 3.31which shows that it was reduced at the mercury electrode producing sharp and characteristic peak at Ep of -1.49 V. The result of voltammetric study of coumarin shows that it is solely involved in the reduction of all studied aflatoxins. As electrochemical behaviour of the aflatoxins closely parallels that of coumarin, it is likely that the electroactivity of these compounds is associated with coumarin moiety in their molecules structure. The main difference in behaviour lies in the fact that the reduction of the aflatoxins occurs at a potential of about 300 – 400 mV more positive than that for coumarin. This can be explained by the increased conjugation caused by ketone group in the neighbouring cyclopentanone (AFB1 and AFB2) or δ-lactone (AFG1 and AFG2) rings. Based on this reason, aflatoxins are easier to reduce compared to coumarin and their Ep are located at more positive than that for coumarin.

Figure 3.31

Voltammograms of coumarin at concentration of (b) 34 µM, (c) 68 µM,

(d) 102 µM, (e) 136 µM and (f) 170 µM in (a) BRB at pH 9.0

114 Based on all previous results, it can be concluded that a double bond in the benzene ring which conjugates with the ketone group undergoes a reduction reaction that produces a single cathodic peak for all studied aflatoxins. This finding is also in agreement with a few reports that have been previously published for compounds that have a double bond in the benzene ring which has conjugated with the ketone group reduced at the mercury electrode. For example, cortisone and testosterone were reduced and peaks were obtained for the reduction of the C = C double bond at -1.77 V and 1.84 V (versus SCE) respectively in 0.1 M tri-ethyl ammonium per chlorate (TEAP)– 50 % methanol as reported by Smyth and Smyth (1978). Both compounds are 3-keto steroids with un-saturation between C4 and C5 as shown in Figure 3.32a and 3.32b.

CH3

O

OH

HO O

CH3

HO O

O

(a) Figure 3.32

(b) Chemical structures of (a) cortisone and (b) testosterone.

Smyth and Smyth (1978) also reported that cardiovascular drugs such as digoxin and digitoxin (Figure 3.33) also undergo reduction reaction which occurs at C=C bond in the five membered ring structure containing the conjugated carbonyl group. Using DPP technique, digoxin and digitoxin gave peak at -2.285 V and -2.325 V (versus SCE) respectively in propan-2-ol – 0.01 M tetrabutylammonium bromide (TBAB).

115 O OH

CH 3

O CH3

O

CH 3

CH3

OH

(C 18H 31O 9 )O

OH

(C18H31O9)O

H

(a) Figure 3.33

O

H

(b)

Chemical structures of (a) digoxin and (b) digitoxin.

Other examples for the compounds that undergo reduction reaction which occurs at C=C bond in their chemical structures are listed in Table 3.2. Other compounds that have the same chemical structure and reduced at the mercury electrode produce cathodic peak are enoxacin (Zhang et al. 1996), ofloxacin (Rizk et al. 1998) and Alizarin Red S (ARS) (Sun and Jiao, 2002). Based on the previous reports, taking into account that the molecular structure of AFB2 is similar in case where the carbonyl group conjugates to the double bond in the benzene ring, it is assumed that AFB2 undergoes reduction reaction at the mercury electrode whereby C=C bond in its chemical structure is reduced at the mercury electrode. The Ep of AFB2 is also pH dependent and shifted towards a more negative direction in increasing pH of the BRB solution which indicates the involvement of protons in the AFB2 electrode reaction (Ghoneim et al., 2003a and Rodriguez et al., 2004). A plot of Ep versus pH of supporting electrolyte is illustrated in Figure 4.34. Two ranges were observed in the Ep – pH plot (Range I for pH from 5 to 8 and Range II for pH from 9 to 12 as in Figure 4.33) with an intersection point at pH 8.7 which was thought to correspond to the pKa value of the adsorbed AFB2 (Navalion et al. 2002). This result again suggests that the deprotonated form of the AFB2 is the electroactive species adsorbed in the HMDE.

116 Compounds reduced at the mercury electrode

Table 3.2

Compound

Ep (V)

Chemical structure

Supporting

Reference

electrolyte NHCH2N(CH2CH3)2

O

Imidazoacridinone

-1.41 N

O

Cakala et

buffer at

al., (1999)

pH 7.4

N

Warfarin

Phosphate

O

-1.40

H

BRB at

Ghoneim

pH 5.0

and Tawfik, (2004)

ONa

CH2COCH3

HO CCH

Lavonogestrel

-1.44

BRB at

Ghoneim et

pH 3.0

al., (2004a)

BRB at

Ibrahim et

pH 5.0

al., (2002)

O

C2H5

Nalidixic acid

N

-1.30

N

H3C

COOH O

117

1250 E p (-m V )

Range II

1200 Range I

1150 1100 4

6

8

10

12

14

pH

Figure 3.34

Effect of pH of BRB solution on the Ep for AFB2. AFB2 concentration:

2.0 µM, Ei = 0, Ef = -1.5 V, Eacc = 0, tacc = 30s, υ = 50 mV/s.

The relation between Ep and pH of the medium over the range, 5.0 to 8.0 is expressed by the following equation: Ep (-mV) =

21.4 pH + 1015.9

(R2 = 0.9777, n = 4)

(3.5)

The Ep remains constant for pH 10.0 to pH 12.0 which suggests that within this range of pH, the reaction is independent of the number of protons involved in the reduction process. 3.2.1.2

Optimisation of Instrumental Conditions

The voltammetric determination of analyte at trace level normally involves very small current responses. For that reason it is important to optimise all the parameters which may have an influence on the measured current. From the previous experiment, BRB solution at pH 9.0 was chosen as the best supporting electrolyte, thus this solution has been used throughout this optimisation procedure using differential pulse cathodic

118 stripping voltammetry (DPCSV) technique. The effect of Ei, Ef, Eacc, tacc, υ and pulse amplitude to the Ip and Ep and of AFB2 peak have been studied. This optimisation procedure used two different concentrations of AFB2 which are 2.0 µM and 0.06 µM for high and low concentrations respectively. The former has been initially used followed with the latter as the final optimisation step. The initial parameters used for this optimisation were; Ei = 0 mV, Ef = -1500 mV, Eacc= 0 mV, tacc = 15 s, υ = 20 mV/s, pulse amplitude = 100 mV and quiescent time = 10 sec. Using all the stated parameters, the Ip obtained for 2.0 µM of AFB2 was 29 nA and the Ep was -1.240 V. 3.2.1.2a

Effect of Scan Rate (υ)

The dependence of Ip and Ep of AFB2 on υ has been studied. The υ was varied from 20 to 100 mV/s while other parameters were unchanged. Figure 3.35a shows a maximun

50

1320

40 30

1290

Ep (-m V)

Ip (n A )

response (36 nA) obtained at scan rate of 40 mV/s.

20 10

1260

0 0

20

40

60

80

Scan rate (mV/s)

(a) Figure 3.35

100

120

1230 0

30

60

90

120

Scan rate (mV/s)

(b)

Effect of various υ to the (a) Ip and (b) Ep of 2.0 µM AFB2 peak in

BRB pH 9.0. Ei = 0, Ef = -1.50 V, Eacc = 0, tacc = 15 s and pulse amplitude = 100 mV. Ip decreases slowly for the increasing υ from 50 to 80 mV/s and sharply drops for 100 mV/s. The increasing υ has shifted the Ep of AFB2 towards a more negative direction according to the equation;

119 E (-mV) = 0.6x (mV/s) + 1230.4

(R2 = 0.9896,

n = 5)

(3.6)

as shown in Figure 3.35b. From this study, the υ of 40 mV/s was used for further optimisation step. 3.2.1.2b

Effect of Accumulation time (tacc)

The effect of tacc to the Ip and Ep of AFB2 peak have been studied over the range of 15 to 60 s as shown in Figure 3.36. It was found that, for the first 40 s, the Ip increases with tacc. This is due to the larger amount of AFB2 which has accumulated at the electrode surface with longer tacc. Since the amount of AFB2 is proportional to the tacc, the resulting peak currents yield a straight line when plotted against the tacc within the stated accumulation range as shown in Figure 3.36a. This relationship follows the equation; Ip (nA) = 0.4895x (x 10-6 M) + 26.132

E p (-m V )

Ip (n A )

60 40 20 0 0

20

40

Accumulation time (s)

(a) Figure 3.36

60

(R2 = 0.9727, n = 5)

(3.7)

1280 1270 1260 1250 1240 1230 0

20

40

60

Accumulation time (s)

(b)

Effect of tacc on (a) Ip and (b) Ep of 2.0 µM AFB2 in BRB at pH 9.0. Ei =

0, Ef = - 1.50 V, Eacc = 0 mV, υ = 40 mV/s and pulse amplitude = 100 mv. However, when the tacc is longer than 40s, the Ip slowly decreases which is due to the complete coverage of the electrode surface (Wang, 1985). This complete coverage

120 phenomenon can be confirmed by increasing drop size of HMDE which resulting increased of the peak height. 3.2.1.2c

Effect of Accumulation Potential (E acc)

The influence of Eacc to the Ip and Ep of AFB2 reduction peak has been observed. An Eacc means the potential at which the AFB2 is deposited at the mercury electrode surface. The optimised Eacc refers to the potential that is the most effective for the deposition of the AFB2 at the mercury electrode surface while AFB2 solution remains unstirred which produces the largest Ip and minimum side reaction. (Wang, 1985). The result shown in Figure 3.37 shows that over a range of 0 to -1200 mV, a highest Ip was found at Eacc of -800 mV which indicates that at this potential, strong adsorption of AFB2 takes place on the electrode surface. The Ip and Ep of AFB2 at this

60 50 40 30 20 10 0

E p (-m V )

Ip (nA )

optimum Eacc are 50 nA and -1.248 V respectively.

0

400

800 Eacc (-mV)

(a) Figure 3.37

1200

1258 1256 1254 1252 1250 1248 1246 0

200 400 600 800 1000 1200 1400 Eacc (-mV)

(b)

The relationship between (a) Ip and (b) Ep with Eacc for 2.0 µM AFB2

in BRB at pH 9.0. Ei = 0, Ef = -1.50 V, tacc = 15 s, υ = 40 mV/s and pulse amplitude = 100 mV. A linear response was obtained for the Eacc over a range of 0 to -0.80 V ( R2 = 0.9769, n = 5). From Figure 3.37b, for Eacc at more negative than -0.80 V with the same Ei (Ei = 0), Ep of AFB2 were constant which indicates that after the maximum Eacc was achieved and the accumulation process of AFB2 at electrode surface was not effective, there are

121 no more changes in Ep. At the same time, Ip decreased until it totally disappeared at Eacc of -1.5 V. The result does not contradict with the suggestion made by Harvey (2000) that the Eacc was usually several tenths of a volt (0.3 to 0.5 V) before the Ep of electroactive species being investigated. Since the highest Ip with no extra peak was obtained at an Eacc of -0.80 V, this potential was chosen as the optimum Eacc. 3.2.1.2d

Effect of Initial Potential (Ei)

Further optimisation was performed by observing the effects of Ei to the Ip and Ep of AFB2. The Ei used in this study were 0, 200, 400, 600, 800 and 1000 mV while Ef was kept constant at -1500 mV. The result is shown in Figure 3.38a. The result indicates that when the Ei was changed to more negative values, the Ip slowly decreased until the Ei was -800mv. However at -1000 mV the Ip sharply increased and gave a maximum response (53 nA). In other words, when the Ei was changed through this manner, the window of scanning potential was smaller and hence, the time for analysis became shorter. For Ei which was more negative than -1000 mV, the scan was not performed due to the very short range of scanning potential. Also because of the instrumental limitations, this experiment cannot be continued unless other instrumental parameters

1260

70 60 50 40 30 20 10 0

E p (-m V )

Ip (nA )

such as pulse amplitude and υ are changed.

1256 1252 1248 1244 1240

0

300

600

900

1200

0

300

Figure 3.38

900

1200

Ei (-mV)

Ei (-mV)

(a)

600

(b)

Effect of Ei on (a) Ip and (b) Ep of 2.0 µM AFB2 in BRB at pH 9.0. Ef = -

1.50 V, Eacc = -0.80 V, tacc = 40 s, υ = 40 mV/s and pulse amplitude = 100 mV

122 From Figure 3.38b, Ep remains constant for Ei of 0 to -400 mV. It started to shift towards a more negative direction when the Ei was -600 mV and remained constant after this value. This experiment reveals that the best potential window was from -1.0 V to -1.50 V which can save the analysis time and at the same time gave maximum Ip. 3.2.1.2e

Effect of Pulse Amplitude

The effect of the pulse amplitude on the Ip shows that the Ip increased and Ep displaced towards less negative direction when the pulse amplitude increased from the range of 30 to 100mV as shown in Figure 4.39. The result shows that a maximum value of Ip (53 nA) was obtained at pulse amplitude of 100 mV. At the higher value of pulse amplitude, the Ip is slightly increased but peak broadening was observed, so 100 mV is

80

1300

60

1260

E p (-m V )

Ip (nA )

chosen for optimum pulse amplitude.

40 20

1220 1180 1140

0 0

30

60

90

120

150

Pulse amplitude (mV)

(a) Figure 3.39

0

30

60

90

120

Pulse amplitude (mV)

(b)

Effect of pulse amplitude on (a) Ip and (b) Ep of 2.0 µM AFB2 in BRB at

pH 9.0. Ei = -1.0 V, Ef = -1.50 V, Eacc = -0.80 V, t acc = 40 s and υ = 40 mV/s. From this study, the optimum conditions for electroanalytical determination of 2.0 µM AFB2 by DPCSV technique were as follows; Ei = -1.0 V, Ef = - 1.5 V, Eacc = -0.80 V, tacc = 40 s, υ = 40 mV/s and pulse amplitude = 100 mV. Using this optimised parameters, the Ip and Ep of 2.0 uM AFB2 were found to be 53 nA and -1.208 V respectively which was able to enhance the peak current to about 83% as compared to that obtained by unoptimised parameters.

123 From the previous optimisation procedures, the Ip of AFB2 was enhanced by 83% and an attempt was made to get higher Ip by further optimisation steps using the lower concentration of AFB2. The reason is using high concentration of AFB2 may lead to the formation of mercury electrode saturation due to the adsorption of AFB2. This optimisation was carried out using 0.06 µM AFB2 and all previous optimised parameters were applied. The optimisation steps followed the same procedure as for 2.0 µM AFB2. The Ip and Ep initially obtained for 0.06 µM AFB2 were 13.90 nA and -1.232 V respectively. The effect of Eacc on the Ip and Ep of AFB2 as shown in Figure 3.40 revealed that the Ip obtained for E acc of -200 mV, -400 mV, -600 mV, -800 mV and -1000mV are not similar which were about 14 nA compared to 11.37 nA and 12.42 nA for 0 and -1100 mV respectively. No peak was observed for Eacc more negative than -1100 mV.

ip (nA)

20 15 10 5 0 0

400

800

1200

1600

Accumulation potential (-mV)

Figure 3.40 Effect of Eacc on Ip of 0.06 µM AFB2. Ei = - 1.0 V, Ef = -1.50 V, tacc = 40

s, scan rate = 40 mV/s and pulse amplitude = 100 mV. Using the potential window of Ei = -1.0 V and Ef = -1.4 V, the optimum Eacc was -0.60 V which gave the Ip of 16.84 nA compared to another potential range (Ei = 1.0 V and Ef = -1.50 V) which produced only 13.78 nA. The Ep of AFB2 remained constant for all studied Eacc. The results suggested that using lower concentration, the optimum Eacc is less negative compared to the higher concentration because for the lower concentration, the electroactive species could easily completely reduce at the mercury electrode. The Ep of AFB2 was independent of the Eacc at the low

124 concentrations. From this experiment, Eacc = -0.6 V, Ei = -1.0 V and Ef = -1.40 V were chosen as the optimum condition for E acc and potential window respectively. The effect of the tacc to the Ip of AFB2 was also investigated and the result is shown in Figure 3.41a which shows that by increasing tacc, for the range of 20 to 80 s, the Ip linearly increased following the equation: (R2 = 0.999, n = 5)

Ip (nA) = 0.4038x (x s) - 0.2847

(3.8)

The maximum Ip of 31.86 nA was obtained at an Eacc of 80 s. When AFB2 was deposited at the mercury electrode for more than 80 s, the Ip was not significantly increased (Figure 3.41a) which may be due to the saturation of the mercury electrode surface and adsorptive equilibrium was achieved (Shams et al. 2004). Due to this reason, the tacc at 80 s was chosen as the optimum tacc . The Ep of AFB2 was shifted

50

1250

40

1240 1230

E p (-m V )

Ip (nA )

toward less negative direction with increasing tacc as shown in Figure 3.41b.

30 20 10

1220 1210 1200

0 0

30

60

90

120

Accumulation time (s)

(a)

150

0

30

60

90

120

150

tacc (s)

(b)

Figure 3.41 Effect of tacc on (a) Ip and (b) Ep of 0.06 µM in BRB at pH 9.0. Ei = - 1.0

V, Ef = -1.40 V, tacc = 80 s, υ = 50 mV/s and pulse amplitude = 100 mV.

The relationship between the υ and Ip of AFB2 cathodic peak was also investigated, which revealed that when the υ is increased, the Ip also increased as shown

125 in Figure 3.42a. For the υ between 20 to 50 mV/s, the Ip is linearly proportional to the υ according to the following equation: (R2 = 0.9999

Ip (nA) = 0.6481x (x mV/ s) + 5. 8407

n = 3)

(3.9)

The Ip was not significantly different when the υ was faster than 60 mV/s. Therefore, 50 mV/s was selected as the optimum υ which gave the maximum Ip of AFB2. The Ep of AFB2 has linearly shifted towards a more negative direction for this range and a small change for the υ of 60 and 80 mV/s as shown in Figure 3.42b. Due to instrumentation limitations, for the υ of 50 mV/s, the pulse amplitude was changed to 80 mV instead of 100 mV. Without that, the Ip of AFB2 could not be automatically calculated by the computer software. The maximum Ip that was obtained from this optimisation was 38.18 nA at Ep of -1.23 V which was about 200% higher than before being optimised (13.90 nA at -1.232 V). Figure 3.44 illustrates the voltammograms of

1225

50 40 30 20 10 0

E p (-m V )

I p (n A )

0.06 uM AFB2 obtained under optimised and unoptimised conditions.

1220 1215 1210 1205 1200

0

20

40

60

80

100

Scan rate (mV/s)

(a)

0

20

40

60

80

100

Scan rate (mV/s)

(b)

Figure 3.42 Effect of υ on (a) Ip and (b) Ep of 0.06 µM AFB2. Ei = - 1.0 V, Ef = -1.40

V, Eacc = -0.60 V and pulse amplitude = 80 mV.

126

Figure 3.43 Voltammogramms of 0.06 µM AFB2 obtained under (a) unoptimised and

(b) optimised parameters in BRB at pH 9.0. From this optimisation steps, the final optimum parameters of DPCSV for determination of 0.06 µM AFB2 compound are; Ei = -1.0 V, Ef = -1.40 V, Eacc = -0.60 V, tacc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV. Table 3.3 represents all optimised parameters for both low and high concentrations of AFB2. Table 3.3

Optimum parameters for 0.06 µM and 2.0 µM AFB2 in BRB at pH 9.0

Parameters

0.06 µM AFB2

2.0 µM AFB2

Ei (V)

-1.0

-1.0

Ef (V)

-1.4

-1.5

Eacc (V)

-0.6

-0.8

tacc (s)

80

40

υ (mV/s)

50

40

Pulse amplitude

80

100

127 For voltammetric study of other aflatoxin except AFG1, the optimum parameters for lower concentration of AFB2 was used throughout the experiments since it give more sensitive and shorter analysis time compared to other optimum parameters. For AFG1, only Ei was changed to -0.95 V to obtain the peak that is located at the middle of the potential range. 3.2.2

Analysis of Aflatoxins

The optimum parameters determined were then used for determination of 0.1 µM AFB2, AFB1, AFG1 and AFG2. The values of Ip and Ep obtained from these analysis are listed in Table 3.4. The relative standard deviation (RSD) for five measurements of each aflatoxins are less than 5% which means that the precision of the method is good and the obtained analytical information has good reliability as reported by Aboul-Eneim et al. (2001). Voltammograms of each aflatoxin are shown in Figure 3.44a to 3.44d. Voltammograms of four aflatoxins which were mixed together are shown in Figure 3.45 which shows that the combination of peak from AFB1 and AFB2 and also from AFG1 and AFG2 produced two shoulders. Table 3.4

The Ip and Ep of aflatoxins obtained by optimised parameters in BRB at

pH 9.0 using DPCSV technique

Aflatoxin, 0.1 µM

Ip (nA), (n=5)

RSD (%)

Ep (V)

AFB1

57.38 ± 0.43

0.75

-1.22

AFB2

57.49 ± 1.02

1.77

-1.23

AFG1

56.85 ± 1.28

2.25

-1.15

AFG2

56.20 ± 1.16

2.06

-1.17

128

(a)

(c) Figure 3.44

(b)

(d)

Voltammograms of 0.1 µM (a) AFB1, (b) AFG1, (c) AFB2 and (d)

AFG2 in BRB at pH 9.0. Ei = -1.0 V (except for AFG1 = -0.95 V), Ef = -1.4 V, Eacc = -0.6 V, tacc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV.

129

Figure 3.45

Voltammograms of (b) mixed aflatoxins in BRB at pH 9.0 as the blank

(a). Parameters condition; Ei = -0.95 V, Ef = -1.4 V, Eacc = -0.6 V, tacc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV. A study was carried out to observe a relationship between Ip and concentration of aflatoxins. For each aflatoxin, the calibration curve was prepared by a series of standard addition of aflatoxins. Statistical parameters such as linearity range, R2 value, limit of detection (LOD), limit of quantification (LOQ), accuracy and precision were evaluated. For ruggedness and robustness tests of the proposed technique, only AFB2 was used. 3.2.2.1 Calibration Curves of Aflatoxins and Validation of the Proposed Method 3.2.2.1a

Calibration curve of AFB2

The calibration curve for AFB2 determination was established by applying the developed procedure. The Ip of AFB2 with increasing concentration is shown in Figure 3.46 which shows that after Ip achieved its maximum value it tends to level off with further addition of AFB2 standard solution as expected for a process that was limited by adsorption of analyte. This phenomenon was similarly observed in the previous study

130 carried out by the CV technique. The linear plot of Ip versus the concentration of AFB2 is shown in Figure 3.47. Their voltammograms are shown in Appendix L

250

Ip (nA)

200 150 100 50 0 0

10

20

30

40

50

-8

[AFB2] x 10 M

Figure 3.46

Increasing concentration of AFB2 in BRB at pH 9.0. The parameter

conditions are Ei = -1.0 V, Ef = -1.4 V, Eacc = -0.60 V, tacc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV.

200

Ip (nA)

160 120 80 y = 5.5385x + 3.2224

40

2

R = 0.9980

0 0

10

20

30

40

-8

[AFB2] X10 M

Figure 3.47

Linear plot of Ip versus concentration of AFB2 in BRB at pH 9.0. The

parameter conditions are the same as in Figure 4.46

It shows that the range of linearity was found to be from 2.0 to 32.0 x 10-8 M (6.29 to 100.63 ppb) with a LOD of 0.796 x 10-8 M (2.50 ppb) and LOQ was 2.65 x 10-8 M (8.33 ppb). The R2 value was 0.9980. The LOD was determined by standard

131 addition of lower concentration of analyte until a sample peak is obtained that was significantly different from the blank signal (Barek et al., 1999). For the determination of precision of the developed method, the reproducibility was calculated from 8 independent measurements of 0.10 µM and 0.20 µM of AFB2 solution on the same day, obtaining relative standard deviations (RSD) of 2.83 % and 0.72% respectively. The precision is good since the mean values do not exceeds 15% of the variance coefficient (CV) (Torreiro et al. 2004). Voltammograms of these measurements are shown in Appendix M. For the inter-days (within 3 days) accuracy determinations, the RSD for day 1, day 2 and day 3 obtained for measurement of 0.10 µM are 2.83%, 2.39% and 1.31% respectively and for 0.20 µM were 0.72 %, 1.03 % and 0.98 %. Voltammograms of these measurements for 0.10 µM AFB2 are shown in Appendix N. The results of these precision studies for intra-day and inter-day are shown in Table 3.5. Table 3.5

Ip (in nA) obtained for intra-day and inter-day precision studies of 0.10

µM and 0.20 µM by the proposed voltammetric procedure (n=8)

[AFB2]

Intra-day measurement Ip ± SD (RSD)

Inter-day measurement Ip ± SD (RSD) Day 1

Day 2

Day 3

0.1 µM

57.68 ± 1.63 (2.83%)

57.68 ± 1.63 (2.83%)

56.52 ± 1.35 (2.39%)

56.59 ± 0.74 (1.31%)

0.2 µM

114.87 ± 0.83 (0.72%)

114.87 ± 0.83 (0.72%)

113.85 ± 1.17 (1.03 %)

112.78 ± 1.11 (0.98%)

132 The accuracy of the method is defined as the closeness of agreement between the experiment result and the true value (Chan et al., 2004). It is determined by calculating the percentage of relative error between the measured mean concentrations and the added concentrations (Torriero, 2004). In order to determine the accuracy of the proposed method, a recovery study was carried out by the addition of an amount of three different concentrations of AFB2 (0.10 µM, 0.15 µM and 0.20 µM) into the voltammetric cell and measuring the peak currents of respective concentrations. The actual amount of AFB2 found in the cell were calculated using the obtained regression equation (y = 5.5385x + 3.2224). The recoveries obtained were 97.29 ± 1.29 %, 97.15 ± 0.79 % and 99.39 ± 0.50 % respectively as shown in Table 3.6. The result shows that the recovery of AFB2 standard is considered good.

Mean values for recovery of AFB2 standard solution (n = 3)

Table 3.6

No of experiment

Amount added (x 10-8 M)

Ip (nA)

Amount found (x 10-8 M)

Recovery (%)

1

10

56.32

9.59

95.90

55.45

9.44

94.40

58.04

9.88

98.80

85.21

14.80

98.67

84.78

14.73

98.25

85.10

14.78

98.53

113.85

19.97

99.85

114.20

20.03

100.15

113.75

19.94

99.70

2

3

15

20

%Recovery ± SD (RSD)

96.37 ± 2.24 (2.39 %)

98.47 ± 0.24 (0.24 %)

99.90 ± 0.23 (0.23 %)

133 The robustness of the proposed technique was evaluated by examining the influence of small variation in some of the most important procedural conditions, including pH (8.5 to 9.5), Eacc (-0.59 V and -0.61 V) and tacc (75 s and 85 s) to the Ip of AFB2 (Ghoneim et al., 2004b). The results of these studies are shown in Table 3.7. Using F test with 95 % degree of freedom, the results showed that none of these variables significantly affected the obtained Ip of AFB2. Thus, the proposed technique could be considered robust. Table 3.7

Influence of small variation in some of the assay conditions of the

proposed procedure on its suitability and sensitivity using 0.10 µM AFB2

Variable

pH

Condition

Ip of AFB2 (nA)

Recovery ±SD (%)

± SD, n=5

n=5

9.0

Eacc = -0.60 V

60.62 ± 0.88

103.01 ±1.50

8.5

tacc = 80 s

58.82 ± 0.79

100.33 ± 1.35

59.30 ± 0.44

101.15 ± 0.75

9.5 Eacc -0.60 V

pH = 9.0

60.62 ± 0.88

103.01 ± 0.48

-0.59 V

tacc = 80 s

58.78 ± 1.02

100.26 ± 1.75

59.82 ± 0.91

102.04 ± 1.56

pH = 9.0

60.62 ± 0.88

103.01 ± 0.48

Eacc = -0.60 V

58.78 ± 0.48

100.26 ± 0.82

60.90 ± 0.76

103.49 ± 1.30

-0.61 V tacc 80 s 75 s 85 s

Moreover, the ruggedness of the proposed technique was examined by applying the procedure to the analysis of 0.1 µM AFB2 using two potentiostat models, BAS CV

134 450 W and VA 757 Metrohm Analysers, under the same optimised experimental parameters. The results of these studies are listed in Table 3.8. Using the same statistical test, the results show no significant difference in the Ip of AFB2 obtained by using both analysers which indicates that the proposed procedure could be considered rugged. Detailed information on F test for robustness and ruggedness evaluations are elaborated in Appendix O. Table 3.8

Results of ruggedness test for proposed method using 0.10 µM AFB2

Ip of AFB2 ± SD

Recovery ± SD (%)

(nA) , n = 5

n=5

BAS CV50W

58.08 ± 1.63

99.11 ± 2.79

VA 757 Metrohm

57.98 ± 0.83

98.94 ± 1.42

Voltammetric analyser

3.2.2.1b

Calibration Curve of AFB1

Using the same approach as carried out for AFB2, the calibration curve for AFB1 was constructed by plot of Ip of AFB1 versus their concentrations. DPCSV measurements of increasing concentrations were applied and the result is shown in Figure 3.48. Figure 3.49 shows the linear curve for AFB1 which has a linear range between 2.0 to 32.0 x 10-8 M (6.29 to 100.63 ppb) with a R2 value of 0.9989. LOD of the measurement of AFB1 was 0.5 x 10-8 M (1.56 ppb). LOQ was 1.67 x 10-8 M (5.2 ppb). Voltammograms of AFB1 with successive standard addition for AFB1 are shown in Appendix P.

135

250

Ip (nA)

200 150 100 50 0 0

10

20

30

40

50

-8

[AFB1] x 10 M

Figure 3.48

Standard addition of AFB1 in BRB at pH 9.0. Ei = -1.0 V, Ef = -1.4 V,

Eacc = -0.60 V, tacc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV.

200

I p (nA)

150 100 y = 5.4363x + 3.7245

50

R2 = 0.9989

0 0

10

20

30

40

[AFB1] x 10-8M

Figure 3.49

Linear plot of Ip versus concentration of AFB1 in BRB at pH 9.0. All

parameter conditions used are the same as stated in Figure 3.48.

136 Repeatability was examined by performing five replicate measurements for 0.10 and 0.20 µM AFB1 both intra-day and inter-day measurements for it precision and the result is shown in Table 3.9. Relative standard deviation (RSD) of 0.10 µM and 0.20 µM for intra-day measurement were 1.83% and 0.74% respectively have been achieved. For inter-day measurements, RSD of 0.10 µM of AFB1 for day 1, day 2 and day 3 were 1.83%, 4.37% and 2.86% respectively and for 0.20 µM RSD of Ip were 0.74%, 1.93% and 0.56%. The results indicate the high precision of the proposed procedure. The accuracy of the proposed method was evaluated by applying the same procedure as for AFB2. The results are shown in Table 3.10 which shows that the accuracy of the proposed method is considered high.

Table 3.9

Ip (in nA) obtained for intra-day and inter-day precision studies of 0.10

µM and 0.20 µM AFB1 by proposed voltammetric procedure (n=5)

[AFB1]

0.10 µM

0.20 µM

Intra-day measurement Ip ± SD (RSD)

Inter-day measurement Ip ± SD (RSD) Day 1

Day 2

Day 3

58.64 ± 1.11

58.64 ± 1.11

58.53 ± 1.01

57.49 ± 0.67

(1.89%)

(1.89%)

(1.72%)

(1.13%)

112.84 ± 0.82

112.84 ± 0.82

112.68 ± 0.81

113.26 ± 0.63

(0.73%)

(0.73%)

(0.72%)

(0.56%)

137 Mean values for recovery of AFB1 standard solution (n = 3)

Table 3.10

No of experiment

Amount added (x 10-8 M)

Ip (nA)

Amount found (x 10-8 M)

Recovery (%)

Recovery ± SD (%) (RSD)

1

10

58.49 57.11 57.80

10.07 9.83 9.95

100.70 98.30 99.50

99.50 ± 1.20 (0.21 %)

2

15

84.70 85.24 85.85

14.89 14.97 15.08

99.27 99.80 100.53

99.87 ± 0.63 (0.63 %)

3

20

112.26 113.32 112.58

19.99 20.06 19.96

99.97 100.31 99.80

100.15 ± 0.49 (0.26 %)

3.2.2.1c

Calibration Curve of AFG1

The validation of this proposed method for quantitative assay of AFG1 was examined via the evaluation of the same parameters as for AFB1. The effect of increasing concentration of AFG1 to their Ip up to 45 x 10-8 M (147.6 ppb) is shown in Figure 3.50. The calibration curve with a linear range of 2.0 to 32.0 x 10-8 M (6.56 to 105 ppb) with a R2 value of 0.9992 was obtained as shown in Figure 3.51. LOD value for this measurement was 1.0 x 10-8 M (3.28 ppb) and LOQ was 3.33 x 10 -8 M (10.93 ppb) Voltammograms of AFG1 with increasing concentration are shown in Appendix Q. The results for the precision and accuracy tests are listed Table 3.11 and 3.12 respectively.

138

250

Ip (nA)

200 150 100 50 0 0

10

20

30

40

50

-8

[AFG1] x 10 M

Figure 3.50

Effect of concentration to Ip of AFG1 in BRB at pH 9.0. Ei = -0.95 V,

Ef = -1.4 V, Eacc = -0.60 V, tacc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV.

200

Ip (nA)

150 100 y = 5.5899x + 3.708

50

R2 = 0.9992

0 0

10

20

30

40

-8

[AFG1] x 10 M

Figure 3.51

Linear plot of Ip versus concentration of AFG1 in BRB at pH 9.0.

The parameter conditions are same as in Figure 3.50.

139 Table 3.11

Ip (in nA) obtained for intra-day and inter-day precision studies of 0.10

µM and 0.20 µM of AFG1 by proposed voltammetric procedure (n=5)

[AFG1]

0.10 µM

0.20 µM

Intra-day measurement Ip ± SD (RSD)

Inter-day measurement Ip ± SD (RSD) Day 1

Day 2

Day 3

58.11 ± 0.93

58.11 ± 0.93

59.34 ± 0.81

57.56 ± 1.09

(1.60%)

(1.60%)

(1.37%)

(1.89%)

117.60 ± 1.24

117.60 ± 1.24

118.85 ± 1.12

117.65 ± 1.21

(1.05%)

(1.05%)

(0.94 %)

(1.02%)

Mean values for recovery of AFG1 standard solution (n = 3)

Table 3.12

No of experiment

Amount added (x 10-8 M)

Ip (nA)

Amount found (x 10 -8 M)

Recovery (%)

Recovery ± SD (%) (RSD)

1

10

58.83 58.16 57.16

9.97 9.98 9.81

99.70 99.80 98.10

99.20 ± 0.95 (0.95 %)

2

15

88.20 89.62 88.78

14.78 15.02 14.88

98.53 100.13 99.20

99.28 ± 0.80 (0.81 %)

3

20

118.16 117.52 118.10

19.80 19.69 19.79

99.00 98.45 98.95

98.80 ± 0.30 (0.30 %)

140 3.2.2.1d

Calibration Curve of AFG2

The applicability of the proposed DPCSV technique as an analytical method for the last type of aflatoxin which is AFG2 was examined by measuring the peak current of AFG2 as a function of its concentration under optimised operational parameters. Figure 3.52 shows the effect of increasing concentration of AFG2 to the Ip. A calibration graph for AFG2 was constructed by standard addition method.

250

Ip (nA)

200 150 100 50 0 0

10

20

30

40

50

[AFG2] / X10-8M

Figure 3.52

Effect of concentration of AFG2 to Ip of AFG2 in BRB at pH 9.0.

Ei = -1.0 V, E f = -1.4 V, Eacc = -0.60 V, tacc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV. The correlation between Ip and concentration of AFG2 was linear within the range 2.0 to 32.0 x 10-8 M (6.60 to 105.61 ppb) as shown in Figure 3.53. The calibration graph was represented by the equation: ip (µA) = 5.7132x (x 10-8 M) + 1.1682; R2 = 0.9989 and n = 10. The LOD was found to be 0.758 x 10-8 M (2.50 ppb) and LOQ was 2.53 x 10-8 M (8.33 ppb). The voltammograms of AFG2 with successive addition of its concentration are shown in Appendix R. The results of precision studies for 0.10 µM and 0.20 µM of AFG2 which were obtained by proposed method are represented in Table 3.13.

141

200

I p (nA)

150 100 y = 5.7132x + 1.1682

50

R2 = 0.9989

0 0

10

20

30

40

-8

[AFG2] x 10 M

Figure 3.53

Linear plot of Ip versus concentration of AFG2 in BRB at pH 9.0. All

parameters used are the same as stated in Figure 3.52. Table 3.13

Ip (in nA) obtained for intra-day and inter-day precision studies of 0.10

µM and 0.20 µM of AFG2 by proposed voltammetric procedure (n=5).

[AFG1]

0.10 µM

0.20 µM

Intra-day measurement Ip ± SD (RSD)

Inter-day measurement Ip ± SD (RSD) Day 1

Day 2

Day 3

58.53 ± 1.05

58.53 ± 1.05

59.11 ± 1.19

58.72 ± 1.14

(1.79%)

(1.79%)

(2.01%)

(1.94%)

115.40 ± 1.69

115.40 ± 1.69

115.20 ± 1.82

115.40 ±1.14

(1.46%)

(1.46%)

(1.58 %)

(0.99%)

For accuracy, it was examined by performing three replicate measurements for 0.1 µM, 0.15 µM and 0.2 µM AFG2 solutions under the same operational parameters. Percentage recoveries (R%) of 100.12 +/- 0.25 %, 99.66 +/- 0.90 % and 100.03 +/- 0.26

142 % were achieved respectively with a mean value of % RSD of 0.47%, which indicates the high accuracy of the proposed technique. Table 3.14 shows the results of accuracy study for AFG2. Mean values for recovery of AFG2 standard solution (n = 3)

Table 3.14

No of experiment

Amount added (x 10-8 M)

Ip (nA)

Amount found (x 10 -8 M)

Recovery (%)

Recovery ± SD (%) (RSD)

1

10

58.14 58.20 59.10

9.97 9.98 10.04

99.97 99.80 100.40

100.12 ± 0.25 (0.25 %)

2

15

86.20 87.50 86.10

14.88 15.10 14.86

99.22 100.70 99.06

99.66 ± 0.90 (0.90 %)

3

20

115.40 115.80 115.20

19.99 20.06 19.96

99.97 100.31 99.80

100.03 ± 0.26 (0.26 %)

The Ip and Ep of the aflatoxins with concentration of 0.10 µM which were obtained by BAS C50W were not significantly different compared to those obtained by VA 757 Metrohm Analyser as shown in Table 3.15, indicating that the proposed technique can be applied using different types of voltammetry analysers. Figures 3.54a to 3.54d show the voltammograms of AFB1, AFB2, AFG1 and AFG2 which were obtained using Metrohm voltammetry analyser. From the validation of the proposed technique, Table 3.16 shows all statistical parameters for the aflatoxins.

143 Table 3.15

Ip and Ep of 0.10 uM aflatoxins obtained by BAS and Metrohm

voltammetry analysers under optimised operational parameters for DPCSV method.

Voltammetry analysers BAS CV 50W

757 VA Metrohm

Aflatoxins Ip (nA) ± SD (RSD)

Ep (V)

Ip (nA) ± SD (RSD)

Ep (V)

AFB1

58.28 ± 2.79 (4.78%)

-1.22

59.88 ± 0.94 (1.57%)

-1.23

AFB2

58.08 ± 1.63 (2.81%)

-1.23

57.98 ± 0.83 (1.43%)

-1.24

AFG1

56.85 ± 1.87 (3.29%)

-1.15

59.26 ± 0.84 (1.42%)

-1.15

AFG2

56.20 ± 1.34 (2.38%)

-1.17

57.56 ± 0.63 (1.10%)

-1.18

3.2.2.2 Determination of Limit of Detection (LOD)

The limit of detection (LOD) is an important quantity in chemical analysis. The LOD is the smallest concentration or amount that can be detected with reasonable certainty for a given analytical procedure (Ahmad, 1993; Miller and Miller, 1993). It is the lowest concentrations that can be distinguished from the noise level as described by Bressole et al. (1996). Thompson (1998) reported that the detection limit caused problems for analytical chemists because they are difficult to interpret and the arbitrary dichotomising of the concentration domain provides misleading viewpoint of the behaviour of analytical systems.

144

Figure 3.54

(a)

(b)

(c)

(d)

Voltammograms of 0.10 µM (a) AFB1, (b) AFB2, (c) AFG1 and (d)

AFG2 in BRB at pH 9.0 obtained by 757 VA Metrohm voltammetry analyser. Ei = -1.0 V (except for AFG1 = -0.95 V), Ef = -1.4 V, Eacc = -0.8 V, tacc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV.

In order to estimate the LOD, some alternative methods have been used to overcome the difficulties such as:

145

Table 3.16

Analytical parameters for calibration curves for AFB1, AFB2, AFG1 and

AFG2 obtained by DPCSV technique using BRB at pH 9.0 as the supporting electrolyte

Aflatoxin

Ep (V)

Regression equations

Sensitivity (µA/µM)

Linearity range (x 10-8 M / ppb)

LOD (x10-8 M / ppb)

R2

B1

-1.22

y = 5.4363x + 3.7245

0.5436

2 – 32 / 6.24 – 99.84

0.5 / 1.56

0.9989

B2

-1.23

y = 5.5385x + 3.2224

0.5539

2 – 32 / 6.29 – 100.63

0.796 / 2.50

0.9980

G1

-1.15

y = 5.5899x + 3.7080

0.5900

2 – 32 / 6.56 – 104.92

1.0 / 3.28

0.9992

G2

-1.17

y = 5.7132x + 1.1682

0.5713

2 – 32 / 6.60 – 105.61

0.758 / 2.50

0.9989

Notes: Ep = Peak potential for aflatoxins at concentration of 0.1 µM y = mx + c;

y = nA, x = x 10-8 M, c = nA

LOD = limit of detection

146 a)

The concentration giving a signal three times the standard error plus the y-intercept divided by the slope of calibration curve (Miller and Miller, 1993; Sturm et al., 1997; Yilmaz et al., 2001: Arranz et al., 2003; Skrzypek et al., 2005 and Nouws et al., 2005). This calculation is used by International Union of Pure and Applied Chemistry (IUPAC) (IUPAC, 1978).

b)

The analyte concentration giving a signal equal to a blank signal plus two or three (Conesa et al., 1996) standard deviations of the blank.

c)

The concentration giving a signal equal to three times (Zhang et al., 1996) or 3.3 times (Perez-Ruiz et al., 2004) the standard deviation of the blank signal divided by the slope from the calibration curve. The latter used 12 blank determinations to calculate standard deviation of the blank.

d)

The concentration giving a signal to noise ratio of 3:1 (3 S/N) (Yardimer and Ozaltin, 2001; Khodari et al., 1997; Wang et al., 1997; Liu et al., 2000; Ibrahim et al., 2002; Al-ghamdie et al., 2004 and Farghaly et al., 2005).

e)

The value of three fold standard deviation from seven determinations of the analyte at the concentration corresponding to the lowest point on the appropriate straight line of the calibration curve which was evaluated by R2 value as reported by Barek et al. (1999).

f)

The concentration giving 3 times standard deviation of intercept of the calibration divided by a slope of the calibration curve as reported by Gao et al. (2004) and El-Desoky and Ghoneim (2005).

147 g)

The lowest concentration of sample that give the significantly different signal from the blank after standard addition of lower concentration (Barek et al., 2001a).

h)

Use of the robust regression method where the detection limit is dealt as a hypothesis test in relation to the presence of analyte in an unknown sample by using the experimental information provided by the calibration set (Ortiz et al., 1993).

The results of the determinations of aflatoxins using several methods according to Barek et al. (2001a), Barek et al. (1999), Zhang et al. (1996) and Miller and Miller (1993) are shown in Table 3.17. These methods were chosen due to its simplicity and are widely used by many researchers. Examples of these calculations for AFB1 are represented in Appendix S, T, U and V for each method respectively. From the results, the LOD values obtained according to Barek et al. (1999), Barek et al. (2001a) and Zhang et al. (1996) are not significantly different as was proven by ANOVA test which is reported in Appendix W (Youmen, 1973). The Method 1 (according to Barek et al., 2001a) was selected for the determination of the LOD of all aflatoxins throughout this study since it gives the lowest LOD value as compared to that obtained by other methods. 3.2.2.3

Determination of Limit of Quantification (LOQ)

The limit of quantification (LOQ) is the lower limit for precise quantitative measurements (Miller and Miller, 1993; Chan et al., 2004). It is determined by the formula of (LOD / 3) x 10 as reported by Ozkan et al. (2003) and Ghoneim et al. (2004). A value of YB + 10SB has been suggested for this limit (Miller and Miller, 1993; Rodriguez et al., 2005) where YB is a blank signal and SB is the standard deviation of blank. The LOQ vaules for all aflatoxins obtained by the proposed methods are listed in Table 3.18.

148

Table 3.17

LOD values for determination of aflatoxins obtained by various methods

Method Aflatoxin

1

2

3

4

x 10-8 M

ppb

x 10-8 M

ppb

x 10-8 M

ppb

x 10-8 M

ppb

B1

0.50

1.56

0.64

2.00

0.75

2.35

1.14

3.56

B2

0.78

2.50

0.89

2.80

0.91

2.86

1.47

4.62

G1

1.00

3.28

1.06

3.50

1.10

3.60

2.09

6.84

G2

0.76

2.50

0.92

3.02

0.86

2.84

2.48

8.18

Notes: Method 1: According to Barek et al. (2001a) Method 2: According to Barek et al. (1999) Method 3: According to Zhang et . (1996) Method 4: According to Miller and Miller (1993)

149

Table 3.18

LOQ values for determination of aflatoxins obtained by various methods

Method Aflatoxin

1

2

3

4

x 10-8 M

ppb

x 10-8 M

ppb

x 10-8 M

ppb

x 10-8 M

ppb

B1

1.67

5.20

2.13

6.67

2.50

7.83

3.80

11.87

B2

2.60

8.33

2.97

9.33

2.03

9.53

4.90

15.40

G1

3.33

10.93

3.53

11.67

3.67

7.87

12.00

22.80

G2

2.53

8.33

3.07

10.07

2.87

9.47

8.27

27.27

Notes: Method 1: According to Barek et al. (2001a) Method 2: According to Barek et al. (1999) Method 3: According to Zhang et al. (1996) Method 4: According to Miller and Miller (1993)

150 3.2.2.4 Interference studies

Possible interference from various metal ions and organic substances were investigated by spiking with excess amount of these interfering substances to the BRB at pH 9.0 containing 0.10 µM of each aflatoxin under optimum experimental conditions. The experiments showed that the presence of up to 10-fold of Al3+, Cu 2+, Ni2+, Pb 2+, Zn 2+, ascorbic acid, β-cyclodextrin and cysteine did not affect much the signal measured which indicates that there was no serious interference to the voltammetric determination of all studied aflatoxins. For example, the effect of Zn ion to the Ip of all aflatoxins is shown in Figure 3.55. Voltammogram of AFB2 with addition of this ion is shown in Figure 3.56(i). The interference study by Zn2+ was further carried out by initially reacted all aflatoxins with Zn2+ in BRB at pH 3.0 before being added into the voltammetric cell containing BRB at pH 9.0. Voltammograms of complex AFB2-Zn with increasing concentration of Zn2+ are shown in Figure 3.56(ii). Figure 3.57 shows the Ip of all aflatoxins which were complexed with Zn2+ decreased drastically with the addition of 1.0 µM Zn2+. This effect indicates that Zn2+ interferes with the measurement of aflatoxins in acid condition.

70 60 50 Ip (nA)

AFB1 40

AFB2

30

AFG1 AFG2

20 [Aflatoxin] = 0.1 uM

10 0 0

0.2

0.4

0.6

0.8

1

1.2

[Zn(II)] / uM

Figure 3.55

Ip of all aflatoxins with increasing concentration of Zn2+ up to 1.0 µM

151

(i) Figure 3.56

(ii)

Voltammograms of (i) 0.10 µM AFB2 and (ii) 0.1 µM AFB2-Zn

complex with increasing concentration of Zn2+ ( a = 0, b = 0.75 µM, c = 1.50 µM, d = 2.25 µM and e = 3.0 µM). Blank = BRB at pH 9.0. Experimental conditions; Ei = -1.0 V, Ef = -1.4 V, Eacc = -0.60 V, tacc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV. . 70 60

Ip (nA)

50

AFB1

40

AFB2

30

AFG1 AFG2

20 10 0 0

1

2

3

4

[Zn] / uM

Figure 3.57

Ip of all aflatoxins-Zn complex after reacting with increasing

concentration of Zn 2+ in BRB at pH 3.0. Measurements were made in BRB at pH 9.0 within 15 minutes of reaction time. Ei = -1.0 (except AFG1 = -0.95 V), Ef = -1.4 V, Eacc = -0.6 V, tacc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV.

152 Similarly, studies carried out using UV-VIS spectrometric technique also showed that absorbance of 1.0 ppm AFB2 decreases with increasing concentration of Zn 2+ (from 0.15 to 0.60 ppm) in acidic condition as shown in Figure 3.58. The same patterns were also found for other aflatoxins. Their absorbances decreased linearly with increasing concentration of Zn2. From this study, it can be concluded that Zn2+ reacted with all aflatoxins in acidic medium but does not in BRB at pH 9.0.

0.12 0.1

Abs

0.08

AFB1 AFB2

0.06

AFG1 AFG2

0.04 0.02 [aflatoxins ] = 1.0 ppm

0 0

0.15

0.3

0.45

0.6

[Zn(II)] / ppm

Figure 3.58

Absorbance of all aflatoxins with increasing concentration of Zn2+ in

BRB at pH 3.0 within 15 minutes of reaction time.

For Zn 2+, studies using initial potential (Ei) at - 0.25 V instead -1.0 V was conducted. The aim was to investigate the response of Zn peak which appeared at -0.98 V with increasing Zn2+ concentration and the result is shown in Figure 3.59. The figure illustrates that the Zn 2+ peak increases while no significant change was observed on Ip and Ep of AFB2 which show that the peak appeared at the Ep of -0.98 was from the Zn 2+.

153

Figure 3.59

Voltammograms of 0.10 µM AFB2 with increasing concentrations of

Zn 2+ (from 0.10 to 0.50 µM) in BRB at pH 9.0. Ei = -0.25 V, Ef = -1.4 V, Eacc = -0.6 V, tacc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV. For interference study by organic substance, ascorbic acid (Figure 3.60) was selected due to its high content in food as a natural micronutrient and plays many physiological roles as reported by Perez-Ruiz et al. (2004). OH

HO

HO

O

Figure 3.60

O

OH

Chemical structure of ascorbic acid

Ascorbic acid may interfere with the measurement of aflatoxins because of its reducing property due to the presence of the ene-1, 2- diol system (Verma et al., 1991). Its structure has a double bond which conjugates to the ketone group, similar to the

154 aflatoxins chemical structures which may reduce at HMDE in BRB at pH 9.0 and interfere the measurement of aflatoxins. The result of this study is shown in Figures 3.61 and 3.62 indicate that no significant change was observed by increasing the concentration of ascorbic acid up to ten-fold of the aflatoxins concentration.

70 60

Ip (nA)

50 40

AFB1 AFB2

30

AFG1

20

AFG2

10 0 0

0.2

0.4

0.6

0.8

1

1.2

[ascorbic a cid] / uM

Figure 3.61 Ip of all aflatoxins with increasing concentration of ascorbic acid up to

1.0 µM. Concentrations of all aflatoxins are 0.1 µM.

Figure 3.62 Voltammograms of 0.1 µM AFB2 with increasing concentration of

ascorbic acid.

155 Interference studies were also carried out with β-cyclodextrin. Cyclodextrins (CDs) are cyclic oligosaccharides with a hydrophilic outer surface and lipophilic central cavity. CDs are formed with a various numbers of glucose units such as α-, β- and γCD, formed by 6, 7 and 8 glucose units, respectively, are present in the greatest amount. The pKa values of α-, β- and γ-CD are 12.33, 12.20 and 12.08, respectively and the species are charged in strong alkaline solution (Fang et al., 1998). In aqueous solutions, CDs are able to solubilise lipophilic moiety of the compound molecule into the central cavity without any covalent bond. Such complexes are called inclusion complexes (Loftson and Brewster, 1996). Due to its inherent interesting inclusion properties which are exhibited both in solid state and in aqueous solution as reported by Fang et al. (1998) and its capability of forming complexes with aflatoxins, β-CD was selected in this study. In optimum pH of supporting electrolyte for the determination of aflatoxins (pH 9.0), the charged β-CD may react with aflatoxins. The result of this study is as in Figure 3.63 and the voltammograms of AFB2 with increasing concentration of β-CD in Figure 3.64 shows that not much interference was observed with increasing concentration of β-CD up to ten-fold of concentration of aflatoxins.

70 60

Ip (nA)

50 AFB1

40

AFB2

30

AFG1 AFG2

20 [Aflatoxins] = 0.1 uM

10 0 0

0.2

0.4

0.6

0.8

1

[B-CD] / uM

Figure 3.63 1.0 µM.

Ip of all aflatoxins with increasing concentration of β- cyclodextrin up to

156

Figure 3.64 Voltammograms of 0.10 µM AFB2 with increasing concentration of β-

cyclodextrin.

Another organic compound which was used in this interference study was Lcysteine (Figure 3.65). O OH H 2N SH

Figure 3.65

Chemical structure of L-cysteine

The result of this study is given in Figure 3.66 which shows that Ip of all aflatoxins decreased slightly when cysteine was added. Cysteine is one of the organic compound that show a high activity for electrocatalytic adsorption on mercury and the most modified electrode as reported by Shahrokhian and Karimi (2004). The result obtained may be due to the accumulation of L-cysteine on the surface electrode as the concentration of cysteine was increased, which made the electrode surface saturated, hence, limiting the adsorption of aflatoxins.

157

70 60

Ip (nA)

50 AFB1

40

AFB2

30

AFG1 AFG2

20 [Aflatoxins] = 0.1 uM

10 0 0

0.2

0.4

0.6

0.8

1

[L-cyste ine ] / uM

Figure 3.66 Ip of all aflatoxins with increasing concentration of cysteine up to 1.0 µM

From this interference study, all studied metal ions and organic compounds neither significantly decreased nor enhanced the Ip of all aflatoxins in BRB at pH 9.0. Attempts have been made to enhance the Ip of aflatoxins by performing mercury electrode modification using poly-L-lysine (PLL) as described by Ferreira et al. (1999). However the aflatoxin peak does not increase and this may be due to unsuitable medium for reaction between aflatoxins and PLL to take place. For example, Ip of AFB2 was enhanced about 15% in BRB at pH 5.0 however at pH 9.0 the Ip was decreased to almost 35% from its original value as shown in Appendix X. 3.3

Square-Wave Stripping Voltammetry (SWSV) of Aflatoxins

Since aflatoxins can be adsorbed on the mercury surface as has been proven by CV and DPCSV studies, another technique of adsorptive stripping method for quantitative measurement of aflatoxins such as SWSV was developed. By using strong adsorptive phenomenon and by accumulation of aflatoxins at a HMDE prior to SWSV measurement, higher sensitivities can be readily achieved in order to obtain a much lower level of detection for aflatoxins. Compared with other pulse techniques, SWSV is faster and the entire potential scan can be completed in approximately 2 s on a single drop of mercury electrode (Jelen et al., 1997). In this study, SWSV was used in combination with an HMDE to study the Ip of aflatoxins in various BRB pH medium using optimum parameters that have been obtained using DPCSV technique. The

158 influences of SWSV instrumental variables such as frequency and voltage step on the SWSV responses have been evaluated. Overall results show that, compared with DPCSV technique, SWSV produces much higher Ip for all aflatoxins at the same concentration. Finally the proposed method was applied to the determination of aflatoxin in groundnut samples. 3.3.1

SWSV Determination of AFB2

Previous optimised parameters that were obtained by DPCSV have been applied to the determination of 0.10 µM AFB2 using SWSV technique which produces a single and well developed peak at Ep of -1.30 V with Ip of 230 nA. The Ip was about 380 % (3.8 times) higher as compared with that obtained by DPCSV which was only 60 nA as shown in Figure 3.67.

Figure 3.67

Voltammograms of 0.10 µM AFB2 obtained by (a) DPCSV and (b)

SWSV techniques in BRB at pH 9.0. Parameters for DPCSV: Ei = -1.0 V, Ef = -1.4 V, Eacc = -0.6 V, tacc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV and for SWSV: Ei = -1.0 V, Ef = -1.4 V, Eacc = -0.6 V, tacc = 80 s, frequency = 50 Hz, voltage step = 0.02 V, amplitude = 50 mV and υ = 1000 mV/s.

159 3.3.1.1 Optimisation of Experimental and Instrumental SWSV Parameters 3.3.1.1a Influence of pH of BRB

Using optimised parameters as stated in Figure 3.67 for SWSV, the pH of BRB was varied between 6.0 and 13.0. Ip of 0.10 µM AFB2 showed maximum value at pH 9.0 as shown in Figure 3.68. This result was the same as found using CV and DPCSV techniques where BRB at pH 9.0 is the best supporting electrolyte for the determination of aflatoxins. Voltammogram of AFB2 in differenct pH is shown in Figure 3.69.

300 250

I p (nA)

200 150 100 50 0 5

6

7

8

9

10

11

12

13

14

pH of BRB

Figure 3.68

Influence of pH of BRB on the Ip of 0.10 µM AFB2 using SWSV

technique. The instrumental parameters are the same as in Figure 3.67.

160

Figure 3.69

Voltammograms of 0.10 µM AFB2 in different pH of BRB. Parameter

conditions; Ei = -1.0 V, E f = -1.4 V, Eacc = -0.6 V, tacc = 80 s, voltage step = 0.02 V, amplitude = 50 mV, frequency = 50 Hz and scan rate = 1000 mV/s 3.3.1.1b Effect of Instrumental Variables

With regard to the instrumental variables, the relationship between Ip and Ei was studied for 0.10 µM AFB2, and Ei was varied from 0 to -1.2 V. It was found that a maximum amount of 250 nA was obtained for Ei = -1.0 as shown in Figure 3.70. The Ip was slowly decreased when the value of Ei was more negative than -1.0 V. .

300

Ip InA)

250 200 150 100 50 0 0

0.2

0.4

0.6

0.8

1

1.2

1.4

- Ei (V)

Figure 3.70

Effect of Ei to the Ip of 0.10 µM AFB2 in BRB at pH 9.0

161 The influence of Eacc to Ip of AFB2 was investigated where the Eacc was varied between 0 to -1.4 V. The maximum value of Ip was obtained at -0.8 V (265 nA) as shown in Figure 3.71. This value was selected for subsequent experiments

300

I p (nA)

250 200 150 100 50 0 0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

- Eacc (V)

Figure 3.71

Effect of Eacc to the Ip of 0.10 µM AFB2 in BRB at pH 9.0

The effect of tacc on the Ip was studied where tacc was varied from 0 to 200 s. The result is shown in Figure 3.72 which reveals that there is linearity up to 100 s according to equation; y = 3.020x + 20.143 (n=6) with R2 = 0.9950, after which it increased slower and levelled off at about 140 s. After 160 s, Ip decreased which may be due to the desorption of the aflatoxins back to the bulk solution. Thus, the tacc of 100 s appeared to be the optimum tacc for the pre-concentration prior to stripping. A linear increase in Ip of AFB2 was observed when frequency was varied from 25 to 125 Hz as shown in Figure 3.73. The frequency value of 125 Hz was adopted as the optimum value.

162

400 350

Ip (nA)

300 250 200 150 100 50 0 0

40

80

120 t

Figure 3.72

acc

160

200

240

(s)

Relationship between Ip of 0.10 µM AFB2 while increasing tacc

1000

Ip (nA)

800 600 400 200 0 0

50

100

150

Frequency (Hz)

Figure 3.73

Effect of frequency to the Ip of 0.10 µM AFB2 in BRB at pH 9.0

The Ip of AFB2 depends linearly on the square root of frequency as shown in Figure 3.74 which indicates that AFB2 adsorbed on the mercury electrode as reported by Komorsky-Lovric et al. (1992). This result is in agreement with that obtained by previous CV and DPCSV experiments.

163

1000

I p (nA)

800 600 400 y = 107.91x - 424.7

200

R2 = 0.9919

0 3

5

7

9

11

(Frequency)1/2

Figure 3.74

Linear relationships between Ip of AFB2 and square root of frequency

The Ip of the AFB2 increases linearly with variation voltage step from 10 mV to 30 mV but after this value the Ip decreases (Figure 3.75). Hence, 30 mV was selected as the optimum voltage step for further experiments.

1000

Ip (nA)

800 600 400 200 0 0

0.01

0.02

0.03

0.04

0.05

Voltage step (V)

Figure 3.75

Influence of square-wave voltage step to Ip of 0.10 µM AFB2.

164 Regarding the influence of the square wave amplitude, the maximum value for Ip was observed at 0.05 V when the amplitude increases from 0.025 to 0.10V as shown in Figure 3.76. The Ep of AFB2 was ideally linear and shifted to a more negative direction with increasing amplitude according to the equation; y = 1.2x -1.36 (n=4, R2 = 1.0) as shown in Figure 3.77. Since the square wave frequency, voltage step and scan rate are interrelated, for frequency of 125 Hz and voltage step of 0.03 V, the scan rate is 3750 mV/ s.

1000

Ip (nA)

750 500 250 0 0

0.025

0.05

0.075

0.1

0.125

Amplitude (V)

Figure 3.76

Influence of square-wave amplitude to Ip of 0.10 µM AFB2.

1.34 1.32 Ep (-V)

1.3 1.28 1.26 y = -1.2x + 1.36

1.24

R2 = 1

1.22 0

0.025

0.05

0.075

0.1

0.125

Amplitude (V)

Figure 3.77

Relationship of SWSV Ep of AFB2 with increasing amplitude

165 Accordingly, the optimum experimental and instrumental parameters of the proposed SWSV procedure for the determination of AFB2 are Ei = -1.0 V, Ef = -1.4 V, Eacc = -0.80 V, tacc = 100 s, frequency = 125 Hz, voltage step = 0.03 V, amplitude = 75 V, scan rate = 3750 mV/s in BRB at pH 9.0 as the supporting electrolyte. Using these parameters, the Ip for 0.10 µM AFB2 was 913 nA with Ep at -1.28 V an increase of 220% before being optimised. Figure 3.78 shows the Ip of 0.10 µM AFB2 obtained under optimised and non-optimised SWSV parameters compared with that obtained under optimised DPCSV parameters.

1050 900

Ip (nA)

750 600 450 300 150 0 a

Figure 3.78

b

c

The Ip of 0.10 µM AFB2 obtained under (a) non-optimised and (b)

optimised SWSV parameters compared with that obtained under (c) optimised DPCSV parameters. The voltammograms of AFB2 are shown in Figure 3.79. As compared to the DPCSV, the Ip value for the optimised parameters for SWSV was 15 times higher. This high sensitivity is one of the SWSV characteristics that was also found by other researchers such as reported by Radi et al. (2001), Guiberteau et al. (2001), El-Hady et al. (2004), Gazy et al. (2004), Farghaly and Ghandour (2005) and Pacheco et al. (2005).

166

Figure 3.79

Voltammograms of 0.10 µM AFB2 obtained under (a) non-optimised

and (b) optimised SWSV parameters compared with that obtained under (c) optimised DPCSV parameters.

3.3.2

SWSV Determination of Other Aflatoxins

Using the optimum SWSV parameters, the Ip of AFB1, AFG1 and AFG2 at concentration of 0.10 µM have been measured in BRB at pH 9.0. The results are shown in Table 3.19. When compared with the Ip that were obtained by DPCSV, overall results show that it increases about 15 times for AFB1 and AFB2 while for AFG1 and AFG2 about 11 times as shown in Figure 3.80. Their voltammograms are shown in Figure 3.81 which also includes DPCSV voltammograms for comparison.

167 Table 3.19

Ip and Ep for all aflatoxins obtained by SWSV in BRB at pH 9.0 (n=5)

Aflatoxin

Ip (nA) / (RSD)

Ep (V)

AFB1

838.8 ± 5.72 (0.68%)

-1.30

AFB2

825.4 ± 11.51 (1.39%)

-1.30

AFG1

633.0 ± 3.16 (0.50%)

-1.24

AFG2

674.2 ± 11.86 (1.76%)

-1.24

1000 800 DPCSV

600 I p (nA)

SWSV

400 200 0

Figure 3.80

AFB1

AFB2

AFG1

AFG2

Ip of 0.10 µM aflatoxins obtained using two different stripping

voltammetric techniques under their optimum parameter conditions in BRB at pH 9.0.

168

(i)

(iii) Figure 3.81

(ii)

(iv)

Voltammograms of (i) AFB1, (ii) AFB2, (iii) AFG1 and (iv) AFG2

obtained by (b) DPCSV compared with that obtained by (c) SWSV in (a) BRB at pH 9.0. 3.3.3

Calibration Curves and Method Validation

After optimisation of the experimental condition, calibration curves were prepared by measuring the Ip of each aflatoxin with different concentration. For example, voltammograms of AFB1 with varying concentrations are shown in Figure 3.82.

169

Figure 3.82

SWSV voltammograms for different concentrations of AFB1 in BRB at

pH 9.0. The broken line represents the blank; (a) 0.01 µM, (b) 0.025 µM, (c) 0.05 µM, (d) 0.075 µM, (e) 0.10 µM, (f) 0.125 µM and (g) 0.150 µM. Parameter conditions; Ei = -1.0 V, Ef = -1.4 V, Eacc = -0.8 V, tacc = 100 s, frequency = 125 Hz, voltage step = 0.03 V, pulse amplitude = 75 mV and scan rate = 3750 mV/s. The calibration curves for all aflatoxins are shown in Figures 3.83 to 3.86. The LOD were determined by standard addition of low concentration of analyte (AFB1, AFB2, AFG1 and AFG2) until a response was obtained that is significantly different from the blank sample (Barek et al., 2001a). LOQ were calculated using the formula LOD/3 x 10. Figure 3.87 shows the comparison of the LOD values obtained by SWSV compared with those obtained by DPCSV. The obtained LOD and LOQ for all aflatoxins are lower than those obtained by DPCSV technique which suggests that the SWSV is much better than DPCSV in terms of sensitivity.

170

1200

Ip (nA)

1000 800 600 y = 70.97x + 76.588 R2 = 0.9984

400 200 0 0

5

10

15 -8

[AFB1] x 10

20

M

Calibration curve for AFB1 obtained by SWSV method.

Figure 3.83

1200

Ip (nA)

1000 800 600 400

y = 61.97x + 243.36

200

R2 = 0.9938

0 0

2.5

5

7.5

10 -8

[AFB2] x 10

12.5

15

M

Calibration curve for AFB2 obtained by SWSV method

Figure 3.84 1000

Ip (nA)

800 600 400 y = 51.046x + 164.79

200

R2 = 0.9912

0 0

5

10

15

[AFG1] x 10-8M

Figure 3.85

Calibration curve for AFG1 obtained by SWSV method.

171

1000

Ip (nA)

800 600 400 y = 49.816x + 157.18

200

R2 = 0.9906

0 0

5

10

15

-8

[AFG2] x 10 M

Figure 3.86

Calibration curve for AFG2 obtained by SWSV method.

3.5

LOD (ppb)

3 2.5 2

DPCSV

1.5

SqWSV

1 0.5 0 AFB1

Figure 3.87

AFB2

AFG1

AFG2

LOD for determination of aflatoxins obtained by two different stripping

methods. Furthermore, this technique provides a shorter time analysis due to the fast scan rate (3750 mV/s). Table 3.20 summarises the characteristics of the calibration plots established using optimum SWSV parameters for all aflatoxins.

172

Table 3.20 Analytical parameters for calibration curves for AFB1, AFB2, AFG1 and AFG2 obtained by SWSV technique in BRB at pH 9.0 as the supporting electrolyte.

Aflatoxin

Ep (V)

Regression Equations

Sensitivity (µA/µM)

Linearity range (x 10-8 M / ppb)

LOD (x 10-8 M / ppt)

LOQ (x 10-8 M/ ppb)

R2

B1

-1.30

y = 70.97x + 76.59

7.097

1 – 15 / 3.12 – 46.73

0.125 / 393

0.42 / 1.31

0.9984

B2

-1.30

y = 61.97x + 243.36

6.197

1– 12.5 / 3.14 – 39.31 0.15 / 468

0.50 / 1.56

0.9938

G1

-1.24

y = 51.046x + 64.79

5.105

1– 12.5 / 3.28 – 40.98 0.15 / 492

0.50 / 1.64

0.9912

G2

-1.24

y = 49.816x + 57.18

4.982

1– 12.5 / 3.30 – 41.25 0.15 / 500

0.50 / 1.67

0.9906

Notes; Ep = Peak potential for aflatoxins at concentration of 0.1 µM y = mx + c;

y = nA, x = x 10-8 M, c = nA

LOD = limit of detection LOQ = limit of quantification

173 Reproducibility was evaluated by performing ten measurements of aflatoxins with concentration of 0.10 µM on the same day and within 3 days for intra and interdays measurements respectively. The results are shown in Table 3.21. For all measurements of all aflatoxins, the RSD were between 0.30% to 1.92% which indicates that the proposed method is reproducible and gives high precision. Voltammograms of 0.1 µM AFB1, AFB2, AFG1 and AFG2 for intra-day precision (n=10) are shown in Appendix Y. Table 3.21

Result of reproducibility study for 0.1 µM aflatoxins in BRB at pH 9.0

Aflatoxins

Ip (nA) for intra-day ( ± SD, n = 10) (RSD)

Ip (nA) for inter-day (± SD, n = 10) (RSD) Day 1

Day 2

Day 3

AFB1

833 ± 10.02 (1.20%)

832 ± 8.77 (1.05%)

833 ± 10.87 (1.30)

833 ± 9.07 (1.09)

AFB2

816 ± 14.88 (1.82%)

817 ± 14.24 (1.74%)

812 ± 15.62 (1.92%)

816 ± 13.92 (1.71%)

AFG1

630 ± 4.38 (0.70%)

634 ± 2.07 (0.33%)

628 ± 2.55 (0.41%)

631 ± 4.32 (0.68%)

AFG2

669 ± 9.92 (1.48%)

674 ± 11.86 (1.76%)

665 ± 4.83 (0.73%)

674 ± 8.41 (1.25)

For assessing accuracy of the proposed method, an amount of 5 x 10 -8 M and 10 x 10-8 M of each aflatoxin were spiked into voltammetric cell and their respective Ip were measured. From the obtained Ip, the actual concentration found was calculated according to the respective calibration plot. The results are summarised in Table 3.22 which show that the recoveries for 5 x 10-8 M and 10 x 10-8 M of all aflatoxins are between 93 to 106% which indicate that the proposed method is of a very high accuracy. Voltammograms of 10 x 10 -8 M of all aflatoxins (n= 3) are shown in

174 Appendix Z. From the method validation, the results show that the proposed SWSV method is satisfactorily precise and accurate, gives very low detection limit and provides very short time analysis. This method was applied for analysis of aflatoxin content in groundnut samples together with the DPCSV method.

Table 3.22

Application of the proposed method in evaluation of accuracy of the

SWSV method by spiking the aflatoxin standard solutions.

Aflatoxin

Amount added ( x 10 -8 M)

Obtained Ip (nA) (± SD, n =3)

Amount found ( x 10 -8 M) (± SD)

Recovery ± SD (%)

RSD of recovery (%)

AFB1

5.0

442.33 ± 2.56

5.15 ± 0.03

103.00 ± 0.60

0.58

10.0

839.00 ± 5.29

10.60 ± 0.07

106.00 ± 0.70

0.66

5.0

566.30 ± 3.79

5.19 ± 0.03

103.64 ± 0.58

0.56

10.0

820.67 ± 6.81

9.39 ± 0.07

93.90 ± 0.70

0.75

5.0

424.33 ± 5.03

5.04 ± 0.06

100.87 ± 1.20

1.19

10.0

639.07 ± 5.51

9.57 ± 0.80

95.70 ± 0.80

0.84

5.0

417.33 ± 2.08

5.24 ± 0.03

104.80 ± 0.53

0.51

10.0

664.00 ± 1.00

10.17 ± 0.02

101.70 ± 0.20

0.20

AFB2

AFG1

AFG2

175 3.4 Stability Studies of Aflatoxins 3.4.1

10 ppm Aflatoxin Stock Solutions

10 ppm aflatoxin stock solutions which were prepared in benzene: acetonitrile (98:2) and stored in the dark under refrigeration at temperature of 14 0 C were monitored for their stability by measuring the absorbance using UV-VIS spectrophotometer every month for twelve months. This study was carried out to determine the shelf life of aflatoxins stock solution in benzene: acetonitrile kept under the dark and cool conditions. The obtained concentrations of all aflatoxins monitored for the period of twelve months at their respective wavelengths are shown in Table 3.23. UV-VIS spectrums for each freshly prepared AFB1, AFB2, AFG1 and AFG2 are shown in Appendix AA. Table 3.23

Average concentration of all aflatoxins within a year stability studies

Aflatoxin

λ range (nm)

Average concentration within a year ± SD (ppm) (RSD)

AFB1

346 – 348

10.63 ± 0.32 (3.01%)

AFB2

349 – 351

10.73 ± 0.42 (3.91%)

AFG1

355 – 357

10.68 ± 0.44 (4.12%)

AFG2

355 – 357

10.58 ± 0.40 (3.78%)

The results show that within a year, all aflatoxins which were stored in the cool and dark conditions are stable and their concentrations are maintained at 10 ppm with RSD of less than 5.0 %.

176 Figures 3.88 to 3.90 show the UV-VIS spectrums for all aflatoxins at 0, 6 and 12 months storage time respectively. There are no significant changes in wavelengths for maximum absorbance of all aflatoxin within the stipulated period of time.

Figure 3.88

UV-VIS spectrum of 10 ppm of all aflatoxins in benzene: acetonitrile

(98%) at preparation date.

Figure 3.89

UV-VIS spectrum of 10 ppm of all aflatoxins in benzene: acetonitrile

(98%) after 6 months of storage time.

177

Figure 3.90

UV-VIS spectrum of 10 ppm of all aflatoxins in benzene: acetonitrile

(98%) after 12 months of storage time.

The maximum absorbance obtained for all aflatoxins are due to the presence of UV chromophore which is a carbonyl group (C=O) in the aflatoxin molecules. This group has a transition electronic level between non-bonding (n) and Π* antibonding which generally occurs at 250 to 300 nm. Due to the conjugation of carbonyl group with a double bond in benzene ring in the aflatoxin structure, this transition takes place at longer wavelength which is more than 300 nm as stated in Table 3.23 (Fifield and Haines, 2000). The result of this study shows that all aflatoxin stock solutions which were stored in the cool and dark conditions have a shelf life of at least one year and can be used for further voltammetric study The same aflatoxin stock solution was exposed to ambient condition, for example, AFB1, the obtained UV-VIS spectrum is different where the wavelength for the maximum absorbance was shifted from 348 nm to 335 nm as shown in Figure 3.91 indicating that the chemical structure of AFB1 may be damaged and cannot be used for voltammetric study.

178

Figure 3.91

UV-VIS spectrums of 10 ppm AFB1 (a) kept in the cool and dark

conditions and (b) exposed to ambient conditions for 3 days. This can be clearly seen when a lower concentration (1 ppm) of the same solution was studied and compared with a new fresh stock solution of the same concentration (Figure 3.92). The result indicates that aflatoxin stock solutions must be protected from light and kept in cool condition for longer shelf life. The spectrum of AFB1 prepared from damaged stock solution shows extra peak at λ of 330 nm while for AFB1 which was prepared from freshly prepared stock solution has only a single peak at the same λ as obtained for the stock solution.

Figure 3.92

UV-VIS spectrums of 1 ppm AFB1 in BRB solution prepared from (a)

freshly prepared and (b) damaged 10 ppm AFB1 stock solution

179 The diluted solutions were kept in the dark and cool conditions for 2 weeks and their absorbance were re-measured. The result is shown in Figure 3.93 which shows that AFB1 from damaged stock produced lower absorbance and at the same time, λ was shifted to lower value from 335 nm to 320 nm compared to the result that was obtained at the preparation date while the AFB1 from freshly prepared stock shows no significant change in its absorbance and λ values. These results indicate that AFB1 solution in BRB prepared from damaged stock is less stable compared to that one prepared from the fresh stock solution.

Figure 3.93

UV-VIS spectrums of 1 ppm AFB1 in BRB solution prepared from (a)

freshly prepared and (b) damaged 10 ppm AFB1 stock solution after 2 weeks of storage in the dark and cool conditions. 3.4.2

1 ppm Aflatoxins in BRB at pH 9.0

4.4.2.1 Month to Month Stability Studies

1.0 ppm aflatoxins prepared in BRB at pH 9.0 which were stored in the freezer at temperature of -4.0 0 C were monitored for their stability within 6 months from the preparation date using the DPCSV technique. The aim of this study was to determine the shelf life of 1.0 ppm aflatoxin in BRB at pH 9.0 kept under the cool and dark conditions. 0.1 µM of each aflatoxin was spiked in 10 ml BRB at pH 9.0 as the

180 supporting electrolyte and the Ip and Ep of respective aflatoxin were observed from 0 to 6 month storage time. The result is shown in Figure 3.94. Voltammograms of all aflatoxins obtained after 0 to 6 month storage time are shown in Appendix AB. The result shows that after three months storage time, the Ip of AFG1 and AFG2 decreased to about 60 % from original Ip measured at first day of preparation while the Ip of AFB1 and AFB2 still maintained about 75% of their respective Ip. Based of this result, the stability order of these aflatoxins is suggested as in the order of AFB2 > AFB1 > AFG2 > AFG1. Based on their chemical structures, aflatoxin with a double bond in the terminal furan ring is less stable in BRB at pH 9.0 as compared to aflatoxin that has a single bond in their furan ring. The finding also reveals that AFG1 and AFG2 can be used up to 2 months while AFB1 and AFB2 up to 3 months. After these periods of time, the respective aflatoxin should be disposed and the new standard solution should be freshly prepared for the rest of experiment.

Percentage (%)

100 80 AFB1 60

AFB2

40

AFG1 AFG2

20 0 0

1

2

3

4

5

6

7

Storage time in freezer (months)

Figure 3.94

Percentage of Ip of 0.1 µM aflatoxins in BRB at pH 9.0 at different

storage time in the cool and dark conditions.

181 4.4.2.2

Hour to Hour Stability Studies

Each aflatoxin (0.1 µM) was spiked in BRB at pH 9.0 and monitored by the DPCSV technique from 0 to 8 hours exposure to ambient conditions. The purpose of this study was to evaluate the stability of aflatoxins in BRB under ambient conditions up to 8 hours. The period of 8 hours was selected due to the normal working hours. A plot of percentage of all aflatoxins versus exposure time is illustrated in Figure 3.95. Voltammograms of all aflatoxins which were exposed from 0 to 8 hours in BRB at pH 9.0 are shown in Appendix AC.

Percentage (%)

100 80 AFB1 60

AFB2

40

AFG1 AFG2

20 0 0

1

2

3

4

5

6

7

8

9

Exposure time (hrs)

Figure 3.95

Percentage of Ip of all aflatoxins in BRB at pH 9.0 exposed to ambient

condition up to 8 hours of exposure time. The result shows that AFB1 and AFB2 measured in BRB at pH 9.0 were stable within 8 hours of exposure time where the Ip for both aflatoxins were reduced only 15% from initial Ip. In contrast, AFG1 and AFG2 gave significant reduction in the Ip within the same period of exposure time where the Ip of AFG1 and AFG2 were reduced to 38% and 48% respectively. The Ep of all aflatoxins were shifted to more negative direction with increasing exposure time as shown in Appendix AC. The experiment reveals that AFG1 and AFG1 were less stable in BRB at pH 9.0 as compared to AFB1 and AFB2.

182 Based on their chemical structures, the most reactive functional groups susceptible for ease of attack by chemical reagents are the two lactones rings of AFG1 and AFG2 and cyclopentanone rings of AFB1 and AFB2. AFG1 and AFG2 were less stable due to the easier opening of the lactone rings in strong alkali as compared to the cyclopentanone ring as reported by Jaimez et al. (2000). The other functional group of this aflatoxin is less readily attacked by any chemical reagents except for the double bond of terminal furan ring in AFB1 and AFG1 which are susceptible to attacks by certain reagents such as TFA (Beebe, 1978; Cespedes and Diaz, 1997; Dhavan and Choudry, 1995; Akiyama et al., 2001; Erdogan, 2004), iodine (Davis and Diener, 1980; Tuinstra and Haasnoot, 1983; Beaver and Wilson, 1990) or bromine in special conditions as shown in Figure 3.96 (Kok et al., 1986). O

O O

HO O

OCH3

O

T FA

O

O 0

30 m in, 50 C

O

I2 O CH3

O

O

O

I

40 s, 60 0 C

O

I

O

OCH3

O O

B r2 4 s , 20 0 C O

Br

O

O

Br OCH3

O O

Figure 3.96

Reactions of 8, 9 double bond furan rings in AFB1 with TFA, iodine

and bromine under special conditions (Kok et al., 1986).

183 As mentioned earlier, 1 ppm AFB1 standard solution from the “damaged” AFB1 stock solution was prepared in BRB at pH 9.0. Beside UV-VIS spectrophotometric determination, it was measured by the DPCSV technique and found that the Ip was only 4.0 nA as compared to the freshly prepared from fresh stock solution which gave a peak current of 60 nA as shown in Figure 3.97. The result indicates that almost all the AFB1 molecules present in BRB solution were already damaged.

Figure 3.97

Voltammograms of 0.1 µM AFB1 obtained in (a) BRB at pH 9.0 which

were prepared from (b) damaged and (c) fresh stock solutions. Another possible reason why aflatoxins were damaged when exposed to ambient condition is due to the fact that aflatoxins are not stable to light. Experiments were performed to verify this statement by running the same experiment as previously mentioned except by wrapping the voltammetric cell with aluminum foil. The results are shown in Figure 3.98 to 3.101 which indicate that in the absence of a lighting system, Ip decreased much faster due to unexplained reason. The result of this experiment shows that the light was not a main factor that caused instability of aflatoxins in BRB at pH 9.0.

184

Ip (nA)

70 60 50

(a)

40 30

(b)

20 10 0 0

2

4

6

8

10

Exposure time (hrs)

Figure 3.98

Ip of 0.1 µM AFB1 obtained in BRB at pH 9.0 from 0 to 8 hours in (a)

light exposed and (b) light protected.

70 60

(a)

Ip (nA)

50

(b)

40 30 20 10 0 0

2

4

6

8

10

Exposure time (hrs)

Figure 3.99

Ip of 0.1 µM AFB2 obtained in BRB at pH 9.0 from 0 to 8 hours in (a)

light exposed and (b) light protected.

185 70 60

Ip (nA)

50 40 (a) (b)

30 20 10 0 0

2

4

6

8

10

Exposure time (hrs)

Figure 3.100 Ip of 0.1 µM AFG1 obtained in BRB at pH 9.0 from 0 to 8 hours in (a)

light exposed and (b) light protected.

70 60

Ip (nA)

50 40 30 (a) (b)

20 10 0 0

2

4

6

8

10

Exposure time (hrs)

Figure 3.101 Ip of 0.1 µM AFG2 obtained in BRB at pH 9.0 from 0 to 8 hours in (a)

light exposed and (b) light protected.

186 3.4.2.3 Stability Studies In Different pH of BRB

0.1 µM of each aflatoxin was measured in four different pH of BRB (6.0, 7.0, 9.0 and 11.0) and the measurements was made in ambient conditions from 0 to 3 hours of exposure time. The purpose of this experiment was to study the stability of all aflatoxins when exposed to ambient condition in slightly acidic, neutral and basic medium in a period of three hours. The results of these studies are shown in Figures 4.103. At 0 hr exposure time, the Ip of all aflatoxins in BRB at pH 6.0 (Figure 3.102a), 7.0 (Figure 3.102b), and 11.0 (Figure 3.102d) were lower as compared in BRB at pH 9.0 (Figure 3.102c) as these pH were not the optimum pH for the voltammetric analysis of aflatoxins. In acidic medium, all aflatoxins were stable within 3 hours exposure time which shows that no reaction has occurred between acid and aflatoxins that can damage the chemical structures of the aflatoxins. This may be due to the formation of the phenolate ions which existed in the resonance state as shown in Figure 3.103 (Heathcote, 1984). However, in strong basic medium (pH 11), Ip for all aflatoxins decreased with longer exposure time and the peak current of AFG1 and AFG2 totally disappeared after 2 and 3 hours respectively. In strong basic medium, hydroxyl ion can slowly react with cyclopentanone and lactone groups of the aflatoxins. AFG1 and AFG2 show faster decreasing value in the Ip compared to AFB1 and AFB2 due to the readiness of the lactone group being attacked in strong basic condition. Figures 3.104, 3.105, 3.106 and 3.107 show the voltammograms of 0.1 µM AFB2 and AFG2 in BRB at pH 6.0 and 11.0 for 0 to 3 hours of exposure time respectively.

187

70

70

pH 6.0

60

50

AFB1

40

AFB2

30

AFG1

I p (nA)

Ip (nA)

50

AFG2

20

AFB1

40

AFB2

30

AFG1 AFG2

20

10

10

0

0 0

1

2

3

0

1

2

3

Exposure time (hrs)

Exposure time (hrs)

(a)

(b)

70

50

pH 9.0

60

pH 11.0

40

50

AFB1

40

AFB2

30

AFG1

20

AFG2

I p (n A)

I p (nA)

pH 7.0

60

AFB1

30

AFB2

20

AFG1 AFG2

10

10

0

0 0

1

2

0

3

1

2

3

Exposure time (hrs)

Exposure time (hrs)

(c)

(d)

Figure 3.102 Peak heights of 0.1 µM aflatoxins in BRB at pH (a) 6.0, (b) 7.0, (c) 9.0 and (d) 11.0 exposed to ambient conditions up to three hours of exposure time.

O

O

O

O_

O

O

OCH3

O

O_

O

OCH3

Figure 3.103 Resonance forms of the phenolate ion (Heathcote, 1984)

OCH3

188

Figure 3.104 Voltammograms of 0.1 µM AFB2 in BRB at pH 6.0 from 0 to 3.0 hrs of exposure time.

Figure 3.105 Voltammograms of 0.10 µM AFB2 in BRB at pH 11.0 (b) from 0 to 3.0 hrs exposure time.

189

Figure 3.106 Voltammograms of 0.10 µM AFG2 in BRB at pH 6.0 from 0 to 3.0 hrs of exposure time.

Figure 3.107 Voltammograms of 0.10 µM AFG2 in BRB at pH 11.0 from 0 to 3.0 hrs of exposure time.

190 The same studies were carried out using UV-VIS spectrophotometer for the same period of exposure time which showed that the absorbance of all aflatoxins in BRB at pH 11.0 decreased with increasing exposure time as shown in Figure 3.108. Overall results are in agreement with that found by voltammetric techniques. UV-VIS

0.16 0.14 0.12 0.1 0.08 0.06 0.04 0.02 0

0.12

pH 6.0

pH 7.0

0.1 AFB1 AFB2 AFG1

AFB1

0.08 Abs

Abs

spectrums for AFB2 and AFG2 in BRB pH at 6.0 and 11.0 are shown in Appendix AD.

AFB2

0.06

AFG1

0.04

AFG2

AFG2

0.02 0 0

1

2

0

3

1

(a) 0.07

pH 11.0

0.06

0.1

AFB1

0.05

AFB1

0.08

AFB2

0.04

AFB2

0.06

AFG1

0.03

AFG1

0.04

AFG2

0.02

AFG2

Abs

Abs

(b)

pH 9.0

0.12

3

Exposure time (hrs)

Exposure time (hrs)

0.14

2

0.02

0.01

0

0 0

1

2

3

Exposure time (hrs)

(c)

0

1

2

3

Exposure time (hrs)

(d)

Figure 3.108 Absorbance of 1.0 ppm aflatoxins in BRB at pH of (a) 6.0, (b) 7.0, (c) 9.0 and (d) 11.0 from 0 to 3 hrs of exposure time.

191

3.4.2.4 Stability Studies in 1.0 M HCl and 1.0 M NaOH The objective of this study was to investigate the stability of all aflatoxins in acid and basic medium within certain period of time where the stability of aflatoxins in 1.0 M HCl and 1.0 M NaOH respectively were carried out. In each case, 0.1 µM of aflatoxin was dissolved in 1.0 M HCl and NaOH respectively before being spiked into the voltammetric cell for voltammetric measurement. The measurement was taken every hour in a period of 6 hours as shown in Figures 3.109 and 3.110. Ip for all the aflatoxins show no significant difference when placed in 1.0 M HCl up to 6 hours as compared to that obtained with 1.0 M NaOH. For aflatoxins left in 1.0 M NaOH, the Ip of AFG1 and AFG2 were significantly reduced as compared to AFB1 and AFB2. The voltammograms of AFB1 and AFG1 in 1.0 M HCl and 1.0 M NaOH for a period of 6 hrs are shown in Appendix AE. No peak was observed after 5 hours of reaction for AFG1 and AFG2. It can be concluded that up to 6 hrs of reaction time, aflatoxins are stable in 1.0 M HCl medium compared with 1.0 M NaOH. This outcome confirmed the results obtained by previous stability studies where all aflatoxins in acidic media are more stable than those in basic media.

80 70

Ip (n A )

60

AFB1

50

AFB2

40

AFG1

30

AFG2

20 10 0 0

1

2

3

4

5

6

Reaction time (hrs)

Figure 3.109 The peak heights of 0.10µM aflatoxins in 1.0 M HCl from 0 to 6 hrs of reaction time.

192

70

Ip (nA)

60 50

AFB1

40

AFB2

30

AFG1

20

AFG2

10 0 0

1

2

3

4

5

6

Reaction time (hrs)

Figure 3.110 The peak heights of 0.10 µM aflatoxins in 1.0 M NaOH from 0 to 6 hrs of reaction time.

3.5

Voltammetric Analysis of Aflatoxins in Real Samples Both the proposed DPCSV and SWSV techniques with their optimum

parameters were applied to the determination of aflatoxins in 15 groundnut real samples. The extraction and clean-up methods for aflatoxin in real samples which have been previously applied by other researchers (Garden et al., 2001; Pena et al., 2002; Chemistry Department, 2002) were tried in order to obtain the highest yield of aflatoxins with the minimum matrix effect. From this study the most reliable technique was chosen for the analysis of the real samples. In addition, a standard addition method was applied for the determination of aflatoxin content in order to overcome any matrix effect (Sanna et al., 2000; Guiberteau et al.,2001; Ghoneim et al., 2003; Rodriguez et

al., 2004). This method also provides the highest precision because the uncertainties introduced by the sample injection are avoided (Chan, 2004). The results which were obtained by these proposed techniques were compared with those obtained by the HPLC which is currently an accepted technique for the routine analysis of aflatoxin compounds in Malaysia (Chemistry Dept., 2000).

193

3.5.1

Study on the Extraction Techniques Three different extraction techniques, labelled as Technique 1, 2 and 3 were

applied in the extraction of groundnut. The results in Figures 3.111a to 3.111c showed the Technique 3 gives a well defined peak current for the aflatoxin as compared to other techniques.

(a)

(b)

(c)

Figure 3.111 Voltammograms of real samples after extraction by Technique (a) 1, (b) 2 and (c) 3 with addition of 10 ppb AFB1 standard solution in BRB at pH 9.0 as a blank.

194 For Technique 3, a standard addition of 10 ppb AFB1 into the sample solution produced a higher Ip of 20.5 nA with the Ep at -1.24 V. However, for the Technique 1, Ip of 10 ppb AFB1 standard solution was only 5 nA and no peak was observed for AFB1 which was spiked in real sample solution obtained by the Technique 2 even though the concentration of standard solution was increased 5 times. This may be due to high matrix effect that eliminates the analyte response. Based on this result, the Technique 3 was selected for extraction and clean-up processes of aflatoxin from groundnut samples throughout this analysis of real samples.

4.5.1

Analysis of Blank The results of these analyses are shown in Figures 3.112a and 3.112b which

show no peak was observed for the blank samples which indicate that the reagents used in extraction and clean-up steps did not produce any peak within the scanned potential range.

(a)

(b)

Figure 3.112 Voltammograms of blank in BRB at pH 9.0 obtained by (a) DPCSV and (b) SWSV methods.

195 Baseline for the blank however, is higher as compared to the BRB at pH 9.0. This may be due to the effect of organic compounds which were used in the extraction process such as methanol and chloroform present in the blank solution which could not be removed completely during nitrogen purging. The voltammetric measurement was repeated after two weeks for the same solution and no significant changes in their voltammograms were observed. Further investigation which involved the addition of 10 ppb AFB2 in the blank sample to observe any effect on Ip of AFB2 was carried out with the DPCSV and SWSV methods. Voltammograms of the blank sample with AFB2 are shown in Figure 3.113 and Table 3.24 shows the Ip of AFB2 for both cases. Since the Ip of 10 ppb AFB2 measured in the presence and absence of the blank sample is not significantly different, it may be concluded that the blank sample does not interfere with the determination of aflatoxin in the real sample.

(a)

(b)

Figure 3.113 DPCSV (a) and SWSV (b) voltammograms of 10 ppb AFB2 (i) in presence of a blank sample (ii) obtained in BRB at pH 9.0 (iii) as the supporting electrolyte.

196

Table 3.24

The Ip and Ep of 10 ppb AFB2 in the presence and absence of a blank

sample

DPCSV (n=3)

SWSV (n=3)

Sample Ip (nA)

Ep (V)

Ip (nA)

Ep (V)

10 ppb AFB2 in BRB at pH 9.0

21.23 ± 0.21 (0.98%)

-1.25

454 ± 2.65 (0.58%)

-1.34

10 ppb AFB2 in BRB at pH 9.0 + blank sample

20.40 ± 0.20 (0.99%)

-1.25

443 ± 3.21 (0.72%)

-1.34

3.5.3

Recovery Studies of Aflatoxins in Real Samples This study was carried out by spiking three different concentration levels (3 ppb,

9 ppb and 15 ppb) into the eluate of ground nut sample followed by the extraction and cleaning-up processes. 15 ppb was the highest concentration used in this study since this is the highest permissible level for total aflatoxin that should be present in groundnut sample as imposed by the Malaysian Government. The voltammetric measurements have been made continuously for three days to observe the recovery of aflatoxins in real samples which was kept in the freezer at 4° C. Figure 3.114 shows the DPCSV voltammograms of real samples added with 3 ppb, 9 ppb and 15 ppb AFB1 in BRB at pH 9.0 for day one. For other aflatoxin such as AFG1 their voltammograms are shown in Appendix AF. The same study was carried out by the SWSV method and for example, the SWSV voltammograms of AFB1 are shown in Appendix AG.

197

(i)

(ii)

(iii)

Figure 3.114 DPCSV voltammograms of real sample (b) added with 3 ppb (i), 9 ppb (ii) and 15 ppb AFB1 (iii) obtained in BRB at pH 9.0 (a) as the blank on the first day of measurement.

198 Figure 3.115 shows the percentage of recovery for all aflatoxins within 3 days obtained by the DPCSV techniques. For those obtained by the SWSV technique, their

120

120

100

100

80

AFB1

% recovery

% recovery

figures are shown in Appendix AH.

AFB2

60

AFG1

40

AFG2

20

80

AFB1 AFB2

60

AFG1

40

AFG2

20

0

0 Day 1

Day 2

Day 3

Day 1

(a)

Day 2

Day 3

(b) 120

% recovery

100 80

AFB1 AFB2

60

AFG1

40

AFG2

20 0 Day 1

Day 2

Day 3

(c)

Figure 3.115 Percentage of recoveries of (a) 3 ppb, (b) 9 ppb and (c) 15 ppb of all aflatoxins in real samples obtained by the DPSV method for one to three days measurements. The results show that at first day of measurement using both techniques, the recoveries of 3 ppb, 9 ppb and 15 ppb of all aflatoxins were from 85 % to 102 % which indicate that the recovery is good. An example on how the recovery is calculated is represented in Appendix AI. The European Committee for Standardisation (CEN Report, 1999) stated that the percentage recovery for aflatoxins in real samples should

199 be 50 to 120%. Based on these criteria, the recoveries of total aflatoxins are acceptable using this developed method. These recovery values slightly decreased with longer keeping time which may be due to the matrix effect. Based on this finding, it is suggested that the recovery study should be performed on the same day of the sample preparation.

3.5.4

Analysis of Aflatoxins in Real Samples In this study, all real samples were analysed using the developed DPCSV and

SWSV methods. For all samples, only the AFB1 was found in this analysis. The DPCSV and SWSV voltammograms of the real samples and with added 10 ppb AFB1 standard solution are shown in Figures 3.116 to 3.121. Examples of real samples with no aflatoxin, small and large traces of aflatoxin are shown in samples S11, S07 and S10 respectively. The DPCSV voltammograms of these samples are shown in Figure 3.116, 3.118 and 3.120 respectively. The sample S11 shows no traces of aflatoxin, S07 contains 5.90 ppb and S10 contains 31.93 ppb aflatoxin. As comparison, the HPLC chromatograms for real samples (S07 and S10) are shown in Appendix AJ.

Figure 3.116 DPCSV voltammograms of real sample, S11 (b) with the addition of 10 ppb AFB1 (c) in BRB at pH 9.0 (a) as the blank. Parameter conditions; Eacc = -0.6 V, tacc = 80 s, scan rate = 50 mV/s and pulse amplitude = 80 mV.

200

Figure 3.117 SWSV voltammograms of real sample S11 (b) with the addition of 10 ppb AFB1 (c) in BRB at pH 9.0 (a) as the blank. Parameter conditions; Eacc = -0.8 V, tacc = 100 s, scan rate = 3750 mV/s, frequency = 125 Hz, voltage step = 0.03 V and pulse amplitude = 75 mV.

Figure 3.118 DPCSV voltammograms of real sample S07 (b) with the addition of 10 ppb AFB1 (c) in BRB at pH 9.0 (a) as the blank. Parameter conditions; Eacc = -0.6 V, tacc = 80 s, scan rate = 50 mV/s and pulse amplitude = 80 mV.

201

Figure 3.119 SWSV voltammograms of real sample S07 (b) with the addition of 10 ppb AFB1 (c) in BRB at pH 9.0 (a) as the blank. Parameter conditions; Eacc = -0.8 V, tacc = 100 s, scan rate = 3750 mV/s, frequency = 125 Hz, voltage step = 0.03 V and pulse amplitude = 75 mV.

Figure 3.120 DPCSV voltammograms of real sample S10 (b) with the addition of 10 ppb AFB1 (c) in BRB at pH 9.0 (a) as the blank. Parameter conditions; Eacc = -0.6 V, tacc = 80 s, scan rate = 50 mV/s and pulse amplitude = 80 mV

202

Figure 3.121 SWSV voltammograms of real sample S10 (b) with the addition of 10 ppb AFB1 (c) in BRB at pH 9.0 (a) as the blank. . Parameter conditions; Eacc = -0.8 V, tacc = 100 s, scan rate = 3750 mV/s, frequency = 125 Hz, voltage step = 0.03 V and pulse amplitude = 75 mV.

The total aflatoxin contents in all 15 samples are shown in Table 3.25. The results that were obtained by the HPLC technique are also listed which are not significantly different. In our study using the both proposed techniques, out of 15 analysed samples, 2 samples (13%) were contaminated with more than 15 ppb of total aflatoxins, 8 samples (53%) were not contaminated and 34% were contaminated with less than 15 ppb of aflatoxin. Higher levels of aflatoxins were observed in groundnuts which have been kept for two weeks in the laboratory (S04 and S10) before being analysed compared to the same samples that were analysed immediately (S01 and S02). This indicates that the storage time also can affect the level of aflatoxins in groundnut samples. Due to that reason other samples were analysed immediately. Appendix AK shows an example on how the aflatoxin content in the S13 was calculated.

203

Table 3.25

Total aflatoxin contents in real samples which were obtained by the

DPCSV, SWSV and HPLC techniques (average of duplicate analysis)

Total aflatoxin (ppb) Samples DPCSV

SWSV

HPLC

S01

12.73

13.92

14.34

S02

7.12

8.36

8.83

S03

n.d

n.d

0.50

S04

21.46

29.72

19.08

S05

9.90

8.95

5.34

S06

n.d

n.d

n.d

S07

5.90

8.17

3.67

S08

n.d

n.d

n.d

S09

n.d

n.d

1.84

S10

31.93

35.65

36.00

S11

n.d

n.d

0.42

S12

n.d

n.d

1.75

S13

10.76

9.21

8.25

S14

n.d

n.d

n.d

S15

n.d

n.d

n.d

n.d: not detected

204

CHAPTER IV

CONCLUSIONS AND RECOMMENDATIONS

4.1

Conclusions The voltammetry technique which is one of the electrochemical methods was

successfully studied through various methods such as cyclic voltammetry (CV), differential pulse cathodic stripping voltammetry (DPCSV) and square-wave stripping voltammetry (SWSV) for determination of aflatoxin compounds. The objectives of this research were to study the electroanalytical properties of aflatoxins and developing voltammetric methods for the determination of these compounds in food samples such as groundnuts. This study met these objectives in terms of the determination of electroanalytical properties of aflatoxins and has also proven the hypothesis regarding these properties for the studied compounds such as the compounds that are actively adsorbed on mercury electrode and undergone totally irreversible reduction reaction. This study was also successful in developing the DPCSV and SWSV methods for qualitative and quantitive determinations of aflatoxins in standard solution and real groundnut samples. The study also proved that aflatoxins which are organic compounds can be determined by voltammetric techniques with convenient quantitation down to 10-9 M (or ppt) concentration level The voltammetric studies of aflatoxins reveals that the proposed DPCSV and SWSV methods are free from inteference effect by studied metal ions and organic compounds. Both techniques are successful in the determination of aflatoxin as

205 individual rather than in mixed condition. An attempt to analyse them simultaneously was not possible due to the fact that the location of the peak potentials of all aflatoxins are very close each other. Harvey (2000) suggested that to get separate peaks for mixed analytes, the difference of their respective peak potential should be more than 0.08 V. All studied aflatoxins (AFB1, AFB2, AFG1 and AFG2) undergo reduction reactions at the mercury electrode with totally irreversible reaction in BRB at pH 9.0. The respective DPCSV and SWSV peak currents increase linearly with increasing concentration for all aflatoxins in the range of 0.02 to 0.32 µM and from 0.01 to 0.125 µM. The peak potentials for all aflatoxins in this solution are located within -1.20 V and they are shifted towards the less negative direction with increasing concentration of aflatoxins. The aflatoxins that are adsorbed on the mercury electrode produce higher peak current with longer accumulation time until a limit is reached due to the saturation of the electrode surface. The DPCSV technique that was successfully developed for the determination of aflatoxins was one which gives a detection limit of around 2.0 ppb for all aflatoxins. This is an acceptable LOD for the determination of aflatoxin in groundnut samples. The optimum parameter conditions for the DPCSV method of the aflatoxins are Ei = 1.0 V (except for AFG1 = -0.95 V), Ef = -1.4 V, Eacc = -0.6 V, tacc = 80 s, scan rate = 50 mV/s and pulse amplitude = 80 mV/s. A SWSV technique was also successfully developed for the analysis of aflatoxins which gives a lower LOD of around 0.5 ppb. The optimum parameter conditions for the SWSV for all aflatoxins are Ei = -1.0 V (except for AFG1 = -0.95 V), Ef = -1.4 V, Eacc = -0.8 V, t acc = 100 s, scan rate = 3750 mV/s, frequency = 125 Hz, voltage step = 0.03 V and pulse amplitude = 50 mV/s. From the statistical analysis of the both proposed techniques, the methods are considered sensitive, accurate, precise, rugged, robust and provide shorter analysis time. From the stability studies of aflatoxins, it can be concluded that aflatoxins stock solution in acetonitrile: benzene (98%) should be kept in the dark and cool conditions for longer shelf life. For aflatoxin standard solutions prepared in BRB at pH 9.0 and

206 stored in the same conditions as for the stock solutions, AFG1 and AFG2 could be used up to 2 months while AFB1 and AFB2 up to 3 months. All afaltoxins are stable in 1.0 M HCl within 6 hours compared to that in 1.0 M NaOH. Both proposed technique are successfully developed and applied in the determination of aflatoxins in groundnuts. The results that were obtained by both techniques are not significantly different than those obtained by the HPLC. As compared to the latter technique, the proposed techniques gave an advantage in terms of analysis time where the voltammetric analysis need less than 2 minutes or even less than 30 s for SWSV technique to complete the analysis of one sample while the HPLC takes approximate 15 min. However, the HPLC is able to separate and analyse all aflatoxins simultaneously compared to the proposed techniques. Finally, it can be concluded that the proposed DPCSV and SWSV methods which are sensitive, accurate, precise, fast, rugged, robust and low cost methods were successfully developed for the determination of aflatoxins in groundnuts samples. Both methods have a great potential as an alternative method for this application in the future.

4.2

Recommendations In this study, the mercury electrode was applied throughout the experiment. Due

to the oxidation of mercury taking place at the oxidation potential of more than 0 V to form mercury (I), the study of aflatoxins were restricted in the range of 0 to -1.5 V. Further study is suggested by using alternative electrodes such as carbon paste, carbon paste with chemical modified electrode, gold, platinum or glassy carbon electrode which has a larger potential window compared to the mercury electrode. Another working electrode such as bismuth film electrode (BFE) can be further investigated for this analysis due to its performance that has been proven to compare favorably to or even surpass to that of mercury electrode (Hutton et al., 2005; Economou, 2005).

207 A study on reagents that can complex with aflatoxins is suggested in order to make the simultaneous determination of aflatoxins in real sample possible. The use of surface active reagents such as alginic acid, Triton X-100 or alkaline phosphatase that can be adsorbed on mercury electrode (Wang, 2000) and inhibit one of the aflatoxins can be studied so as to observe their effect on the peak current of aflatoxins either as in standard solution or simulation samples. A study of the effect of storage condition and the storing time duration of groundnut samples to the level of aflatoxins using the DPCSV and SWSV techniques is suggested. The results from this study can provide valuable information on good practice in storing groundnuts at home and for obtaining a data base regarding the storing time of groundnuts to avoid any contamination by aflatoxins. The stability of aflatoxins which are treated at various temperatures and exposure time to ultra violet (UV) light is suggested. The reason for this study is to determine the degree of temperature and exposure time to UV light that can totally diminish the aflatoxins. Finally, in this study, the DPCSV and SWSV techniques were applied in analysed of groundnut samples but for the future works, both techniques can be explored for analysis of aflatoxins in other types of samples such as rice, cooking oil, cigarettes tobacco, local and imported meats, bread, fish and vegetables due to higher consumption by the Malaysian population. Some reports have been published regarding these analyses (Aycicek et al., 2005; Edinboro and Karnes, 2005; Shenasi and Candlish, 2002; Arrus et al., 2004; AbdulKadar et al., 2004; Erdogan, 2004; Gowda, 2004) but unfortunately none of them are related to the local environment in Malaysia except a paper by Ali et al. (2005) which focused on the analysis of aflatoxins in commercial traditional herbal medicines used in Malaysia and Indonesia.

208

REFERENCES

Abbas, A.N. and Mostafa, G.A.E. (2003). New Triiodomercurate-Modified Carbon Paste Electrode for the Potentiometric Determination of Mercury. Anal. Chim.

Acta . 478:329-335. Abbaspour, A. and Moosavi, S.M.M.(2002). Chemically Modified Carbon Paste Electrode for Determination of Copper (II) by Potentiometric Method. Talanta.

52: 91-96. Abdine, H. and Belal, F. (2002). Polarographic Behaviour and Determination of Acrivastine Capsules and Human Urine. Talanta. 56: 97-104 Abdulkadar, A.H.W., Abdulla, A. A. A., Afrah M. A. and Jassim, H.A. (2004). Mycotoxins in Food Products Available in Qatar. Food Control. 15: 543-548. Aboul-Eneim, H. Y., Stefan, R-I. and Baiulescu, G-E. (2000). Quality and Realibility in

Analytical Chemistry. New York: CRC Press. Abu Zuhri, A. Z., Zatar, N. A., Shubeitah, R.M. and Arafat, H.H. (2000). Voltammetric and Spectrophotometric Determination of Nizatidine in Pharmaceutical Formulations. Mikrochim. Acta.134:153-160. Ahmad, M. (1993). Pengenalan Statistik Dalam Kimia Analisis. Kuala Lumpur: Dewan Bahasa Dan Pustaka. Akiyama, H., Goda, Y. Tanaka. T, and Toyoda, M. (2001). Determination of Aflatoxins B1, B2, G1 and G2 in Spices Using a Multifunctional Column Clean-up, J. of

Chromatography A. 932: 153-157.

209 Alborzi, S., Pourabbas, B., Rashidi, M. and Astaneh, B. (2005). Aflatoxin M1 Contaminated in Pasteurised Milk in Shiraz (south of Iran). Food Control. Article in press. Al-Ghamdi, A.H., Al-shadokhy, M.A. and Al-watan, A.A. (2004). Electrochemical Determination of Cephalothin Antibiotic by Adsorptive Stripping Voltammetric Technique. J.Pharm. Bioanal. Anal. 35(5):1001-1009. Ali, N., Hashim, N.H., Saad, B., Safan, K., Nakajima, M. and Yoshizawa, T. (2005). Evaluation of a Method to Determine the Natural Occurence of Aflatoxins in Commercial Traditional Herbal Medicines From Malaysia and Indonesia. Food

and Chemical Toxicology. 43(12): 1763-1772. Almeida, l.E., Castilho, M., Maza, L.H. and Tabak, M.(1998). Voltammetric and Spectroscopic Studies of the Oxidation of the Anti-oxidant Drug Dipyridamole in Acetonitrile and Ethanol. Anal. Chim. Acta. 375 : 223-231. Ammida, N.H.S., Micheli, L. and Palleschi, G. (2004). Electrochemical Immunosensor for Determination of Aflatoxin B1 inBarley. Anal. Chim. Acta. 520: 159-164. Andrieux, C.P., Hapiot, P. and Saveant, J.M.(1990). Ultramicroelectroded for Fast Electrochemical Kinetics. Electroanalysis. 2:183-193. Androu, V.G., Nikolelis, D. and Tarus, B. (1997). Electrochemistry Investigation of Transduction of Interactions of Alfatoixin M1 With Bilayer Lipid Membranes (BLMs), Anal. Chim. Acta. 350:121-127. Aoki, K. (1990). Theory of Stationary Current Potential Curves at Interdigitated Microarray Electrodes for Quasi-reversible and Totally Irreversible Electrode Reactions. Electroanalysis. 2 : 229-233.

210 Arancibia, V., Alarcon, L. and Sefura, R. (2004). Supercritical Fluid Extraction of Cadmium as Cd-Oxime Complex From Human Hair; Determination of SquareWave Anodic or Adsorptive Stripping Voltammetry. Anal. Chim. Acta. 502 : 189-194. Arranz, A., Dolara, I., de Betono, S.F., Moreda, J. M., Cid, A. and Arranz, J.F. (1999). Electroanalytical Study and Square Wave Voltammetric Techniques for the Determination of β-blocker Timolol at the Mercury Electrode. Anal. Chim

.Acta. 389 : 225-232. Arranz, P., Arranz, A., Moreda, J. M., Cid, A. and Arranz, J.F. (2003). Stripping Voltammetric and Polarographic Techniques for Determination of Anti-fungal Ketoconazole on the Mercury Electrode. J. Pharm. Biomed. Anal. 33: 589-596. Arrigan, D. W. M. (1994). Voltammetric Determination of Trace Metals and Organics After Accumulation at Modified Electrodes. Analyst. 119: 1953-1966. Arrus, K., Blank, G., Abramson, D. Clear, R. and Holley, R. A. (2004). Aflatoxins Production by Aspergillus flavous in Brazil Nuts. J of Stored Products

Research. 41(5): 513-527. Aslanoglu, M. and Ayne, G. (2004). Voltammetric Studies of the Interaction of Quinacrine With DNA. Anal. Bioanal.Chem. 380: 658-663. Aycicek, H., Aksoy, A. and Saygi, S.(2005). Determination of Aflatoxin Levels in Some Dairy and Food Products Which Consumed in Ankara, Turkey. Food

Control. 16: 263-266. Aziz, N.H. and Moussa, L.A., (2002). Influence of Gamma-radiation on Mycotoxin Producing Moulds and Mycotoxins in Fruits. Food Control. 13 : 281-288.

211 Aziz, N.H., Youssef, Y.A., El-Fouly, M.Z. and Moussa, L.A., (1998). Contamination of Some Common Medicinal plant Samples and Spices by Fungi and Their Mycotoxins. Bot. Bull. Acad. Sin. 39: 279-285. Badea, M., Micheli, L., Messia, M. C., Candigliota, T., Marconi, E., Mottram, T., Velasco-Garcia, M., Moscone, E. and Palleschi, G. (2004). Aflatoxin M1 Determination in Raw Milk Using a Flow-injection Immunoassay System. Anal

Chim Acta. 520: 141-148. Bakirci, I. (2001). A Study On the Occurence of Aflatoxin M1 in Milk and Milk Products Produced in Van Province of Turkey. Food Control. 12 : 47-51. Bankole, S.A., Ogunsanwo, B.M. and Eseigbe, D.A. (2005a). Aflatoxins in Nigerian Dry-roasted Groundnuts. Food Chemistry. 89: 503-506. Bankole, S.A., Ogunsanwo, B.M. and Mabekoje, O.O.(2004) Natural Occurrence of Moulds and Aflatoxin B1 in Melon Seed From Markets in Nigeria. Food and

Chemical Toxicology. 42: 1309-1314. Bankole, S.A., Ogunsanwo, B.M., Osho, A. and Adewuyi, G.O. (2005b). Fungal Contamination and Aflatoxin B1 of ‘Egusi’ Melon Seeds in Nigeria. Food

Control. Article in press. Barbeira, P.J.S. and Stradiotto, W.R., (1997). Simultaneous Determination of Trace Amounts of Zinc, Lead and Copper in Rum by Anodic Stripping Voltammetry.

Talanta. 44: 185-188. Barker, G.C. and Jenkins, I.L. (1952). Square-wave Polarography. Analyst. 77 : 685695.

212 Barek, J., Fogg, A. G., Muck, A. and Zima, J. (2001a). Polarography and Voltammetry at Mercury Electrodes. Critical Reviews In Analytical Chemistry.

31: 291-309. Barek, J., Cvacka, J., Muck, A. and Quaiserova, A. (2001b). Electrochemical Methods for Monitoring of Environmental Carcinogens. Fresenius J Anal Chem.

369: 556-562. Barek, J., Pumera, M., Muck. A., Kaderabkova, M. and Zima, J. (1999). Polarographic and Voltammetric Determination of Selected Nitrated Polycyclic Aromatic Hydrocarbons. Anal Chim Acta. 393: 141-146. BAS (1993) Controlled Growth Mercury Electrode Instruction Manual, Bioanalytical System, Inc. Indiana. Batista, L.B., Chalfoun, S.M., Prado, G., Schwan, R.F. and Wheals, A.E. (2003). Toxigenic Fungi Associated With Processed (green) Coffee Beans (Coffea

arabica-L). Int J Of Food Microbiolog. 85 : 293 – 300. Beaver, R.W. and Wilson, D.M. (1990). Comparison of Postcolumn DerivatisationLiquid Chromatography With Thin-layer Chromatography for Determination of Aflatoxins in Naturally Contaminated Corn. J. Assoc.Off. Anal. Chem. 73(4): 379 – 381. Beaver, R.W., James, M.A. and Lin, T.Y. (1991). ELISA-based Screening Test With Liquid Chromatography for the Determination of Aflatoxin in Corn. J.

Assoc.Off. Anal. Chem. 74: 827 – 829. Beebe, B.M. (1978). Reverse Phase High Pressure Liquid Chromatographic Determination of Aflatoxins in Foods. J. Assoc. Off. Anal. Chem. 81 : 13471352.

213 Begum, F. and Samajpati, N. (2000). Mycotoxin Production on Rice, Pulses and Oilseeds. Naturwissenschaften. 87: 275-277. Berzas, J.J., Rodriguez, J., Castaneda, G. and Villasenor, M.J. (2000). Voltammetric Behavior of Sildenafil Citrate (Viagra) Using Square-wave and Adsorptive Stripping Square-wave Techniques. Determination in Pharmaceutical Products. Anal. Chim. Acta. 417: 143-148. Betina, V. (1984). Mycotoxin – Production, Isolation, Separation and Purification. Netherland: Elsevier Science. Bicking, M.K.L., Kniseley, R.N and Svec, H.J. (1983). Coupled-Column for Quantitating Low Levels of Aflatoxins. J . Assoc. Off. Anal. Chem. 66 : 905-908. Bijl, J. and Peteghen, C.V.(1985). Rapid Extraction and Sample Clean-up for the Fluorescence DensitometricDetermination of Aflatoxin M1in Milk and Milk Powder. Anal. Chim. Act. 170: 149-152. Billova, S., Kizek, R., Jelen, F. and Novotna, P. (2003). Square-wave Voltammetric Determination of Cefoperazone in a Bacterial Culture, Pharmaceutical Drug, Milk and Urine. Anal Bioanal Chem. 377: 362-369. Bingham, A.K., Huebner, H.J., Phillips, T.D. and Baver, J.E. (2004). Identification and Reduction of Urinary Aflatoxin Metabolites in Dog. Food and Chemical Toxicology. 42(11): 1851-1858. Blanc, R., Gonzalez-Casado, A., Navabu, A. and Vilchez, J.L. (2000). The Estimate of Blanks in Differential Pulse Voltammetric Technique: Application to Detection Limits Evaluation as Recommended by IUPAC. Anal. Chim. Acta.

403: 117-123.

214 Blesa, J., Soriano, J.M., Molto, J.C., Marin, R. and Manes, J. (2003). Determination of Aflatoxins in Peanuts by Matrix Solid-phase Dispersion and Liquid Chromatography, J of Chromatography A. 1011 : 49-54. Bond, A.M., Oldham, K.B. and Zoski, C.G. (1989). Steady State Voltammetry.

Anal. Chim. Acta. 216: 177-230. Brainina, Kh. (1974). Stripping Voltammetry in Chemical Analysis. New York: Wiley and Sons. Brainina, Kh., Malakhova, N.A. and Stojko, Y. (2000). Stripping Voltammetry in Environmental and Food Analysis. Fresenius J Anal Chem. 368: 307-325. Brera, C., Caputi, R., Miraglia, M., Iavicoli, I., Salerno, A. and Carelli, G. (2002). Exposure Assessment to Mycotoxins in Workplace: Aflatoxins and Ochratoxin A Occurence in Airborne Dust and Human Sera. Microchemical J. 73(1-2): 167173. Bressolle, F., Bromet-Petit, M. and Audron, M. (1996). Validation of Liquid Chromatography and Gas Chromatography Methods: Application to Pharmacokinetic. J. Chromatogr, B. 686 : 3-10. Buffle, J. and Tercier-Waeber, M-L. (2005). Voltammetric Environmental TraceMetal Analysis and Speciation: From Laboratory to in situ Measurements. Trend

In Anal. Chem. 24(3): 172-191. Cakala, M., Mazerska, Z., Donten, M. and Stojek, Z. (1999). Electroanalytical and Acid-base Properties of Imidazoacridinanone, an Antitumor Drug (C-1311).

Anal. Chim. Acta. 379: 209-215.

215 Canabillas, A.G., Burguillos, J.M.O., Canas, M.A.M, Caceres, M.I.R. and Lopez, F.S. (2003). Square Wave Adsorptive Stripping Voltammetric Determination of Piromidic acid. Application in Urine. J Pharm. Biomed. Anal. 33 : 553-562. Carapuca, H.M., Cabral, D.J. and Rocha, L.S. (2005). Adsorptive Stripping Voltammetry of Trimethoprim: Mechanistic Studies and Application to the Fast Determination in Pharmaceutical Suspensions. J Pharm. Biomed. Anal. 38: 364369. Carlson, M.A. , Bargeron, C.B. , Benson, R.C., Fraser, A.B., Phillips, T.E., Velky, J. T., Groopman, J.D., Strickland, P.T. and Ko, H.W. (2000). An Automated, Handheld Biosensor for Aflatoxin. Biosensors and Bioelectronic. 14 : 841-848. Carpini, G., Lucavelli, F., Marrazza, G. and Mascini, M., (2004). OligonucleotideModified Screen-printed Gold Electrodes for Enzyme Amplified Sensing of Nucleic Acids. Biosensors and Bioelectronic. 20: 167-175. Castro, A.A., Wagener, A.L.R., Farias, P.A.M. and Bastos, M. B. (2004). Adsorptive Stripping Voltammetry of 1-hydroxypyrene at the Thin-film Mercury Electrode-basis for Quantitative Determination of PAH Metabolite in Biological Materials. Anal. Chim. Acta. 521 : 201-207. CEN Report. (1999). Food analysis – Biotoxins – Criteria of Analytical Methods of Mycotoxins. European Committee for Standardisation CR 13505:99 E. 1-7. Cepeda, A., Franco, C.M., Fente, C.A., Vazquez, B.I., Rodriguez, J.L., Prognon, P. and Mahuzier, G. (1996). Post-collumn Excitation of Aflatoxins Using Cyclodextrins in Lliquid Chromatography for Food Analysis. J of Chromatogr.

A. 721 : 69-74.

216 Cespedez, A.E and Diaz, G.J. (1997). Analysis of Aflatoxins in Poultry and Pig Feeds and Feedstuffs used in Colombia. J. Assoc. Off. Anal. Chem. 80: 12151219. Chan, C.C. (2004). Potency method validation. In: Chan, C.C., Lam, H., Lee, Y.C. and Zhang, X-M. Analytical Method Validation and Instrument Performance

Verification. USA: John Wiley and Sons. Chan, D., MacDonald, S.J., Boughtflower, V. and Brereton, P. (2004). Simultaneous Determination of Aflatoxins and Ochratoxin A in Food Using a Fully Automated Immunoaffinity Column Clean-up and Liquid ChromatographyFluorescence Detection. J of Chromatoraphy A. 1059 : 13-16. Chang, H.L. and De Vries, J.W. (1983). Rapid High Performance Liquid Chromatography Determination of AFM1 in Milk and Nonfat Dry Milk. J.

Assoc. Off. Anal. Chem. 66 : 913-917. Chemistry Department, Ministry of Science Technology and Innovation. (2000).

Standard Procedure for Determination of Aflatoxins (unpublished paper), Malaysia. Chiavaro, E., Asta, D., Galaverna, G., Biancardi, A., Gambarelli, E., Dossena, A. and Marchelli, R. (2001). New Reversed-phase Liquid Chromatography Method to Detect Aflatoxins in Food and Feed with Cyclodextrins as Fluorescence Enhancers Added to the Eluent. J of Chromatogr. A. 937: 31-40. Chu, F.S., Fan, T.S.L., Zhang, G.S., Xu, Y.S. Faust, S. and Mac Mohan, P.L. (1987). Improved Enzyme-linked Immunosorbent Assay for Aflatoxin B1 in Agricultural Commodities. J. Assoc. Anal. Chem. 70: 854-857.

217 Ciucu, A.A., Neguleseus, C. and Beldwin, R. P. (2003). Detection of Pesticides Using an Amperometric Biosensor Based on Ferophthalocyanine Chemically Modified Carbon Paste Electrode and Immobilised Enzymetic System.

Biosensors and Bioelectronic.18: 303-310. Cole, R.O., Holland, R.D. and Sepaniak, M.J. (1992). Factors Influencing Performance in the Rapid Separation of Aflatoxins by Micellar Electrokinetics Capillary Chromatography. Talanta.39: 1139-1147. Colombo, C. and van den Berg, C.M.G. (1998). In-line Deoxygenated for Flow Analysis With Voltammetric Detection. Anal. Chim. Acta. 377 : 229-240. Conesa, A. J., Pinilla, J. M. and Hernandez, L. (1996). Determination of Metbendazole in Urine by Cathodic Stripping Voltammetry. Anal. Chim. Acta .

331: 111-116. Creepy, E.E. (2002). Update of Survey, Regulation and Toxic Effect of Mycotoxins in Europe. Toxicology Letter. 127: 19-28. Czae, M. Z. and Wang, J. (1999). Pushing the Detectibility of Voltammetry: How Low Can Go ?. Talanta . 50 : 921-928. Dahmen, E.A.M.F. (1986). Electroanalysis: Theory and Application in Aqueous and

Non-aqeuous Media and in Automated Chemical Control. New York: Elsevier. Davies, N.D. and Diener, U.L. (1980). Confirmatory Test for the High Pressure Liquid Chromatographic Determination of Aflatoxin B1. J. Assoc. Off. Anal.

Chem. 63 (1): 107- 109.

218 De Botono, S.F., Moreda, J.M., Arranz, A. and Arranz, J.F. (1996). Study of the Adsorptive Stripping Voltammetric Behaviour of the Antihypertensive Drug Doxazosin. Anal. Chim. Acta . 329: 25-31. De Vries, J. (1996). Food Safety and Toxicity. New York: CRC. Dean, J.A. (1995). Analytical Chemistry Handbook. USA: Mc Graw Hill. Demetriades, D., Economou, A. and Voulgaropoulos, A. (2004). A Study of PencilLead Bismuth-film Electrodes for the Determination of Trace Metals by Anodic Stripping Voltammetry. Anal. Chim. Acta. 519: 167-172. Dhavan, A.S. and Choudry, M.R. (1995). Incidence of Aflatoxins in Animal Feedstuffs: A decade’s Scenario in India. J. AOAC Int. 78 (3): 693 – 698. Dragacci, S., Gleizes, E., Fremy, J.M. and Candlish A.A.G. (1995). Use of Immunoaffinity Chromatography as a Purification Step for the Determination of AFM1 in Cheese. Food Additives and Contaminants. 12(1): 59-65. Duhart, B.T., Shaw, S., Wooley, M., Allen, T. and Grimes, C. ( 1988a). Determination of Aflatoxins B1, B2, G1 and G2 by High Performance Liquid Chromatography With Electrochemical Detector. Anal. Chim. Acta. 208 : 343346. Eaton, D.L and Groopman, J.D. (1994).The Toxicity of Aflatoxin . United Kingdom: Academic Press. Economou, A. (2005). Bismuth-film Electrode: Recent Development and Potentialities for Electroanalysis. Trends Anal. Chem. 24(4): 334-340.

219 Economou, A. and Fielden, P.R. (1995). A Study of Riboflavin Determination by Squarw-Wave Adsorptive Stripping Voltammetry on Mercury Film Electrode, Electroanalysis. 7 : 447 – 453. Economou, A., Bolis, S.D., Efstathiou, C.E. and Volikakis, G.J. (2002). A Virtual Electroanalytical Instrument for Square-wave Voltammetry. Anal. Chim. Acta .

467: 179-188. Edinboro, L.E. and Karnes, H.T. (2005). Determination of Aflatoxin B1 in Sidestream Cigarette Smoke by Immunoaffinity Column Extraction Coupled With Liquid Chromatography/mass Spectrometry. J of Chrom. A. 1083: 127132. Egner, P.A., Munoz, A and Kensler, T.W. (2003). Chemoprevention With Chlorophylin in Individuals Exposed to Dietary Aflatoxins. Mutation Research.

523: 209-216. El-Desoky, H.S. and Ghoneim, M.M. (2005). Assay of the Anti-psychotic Drug Haloperidol in Bulk Form, Pharmaceutical Formulation and Biological Fluids Using Square-wave Adsorptive Stripping Voltammetry at a Mercury Electrode.

J Pharm. Biomed. Anal. 38 : 543-550. El-Hady, D.A., Abdel-Hamid, M.I., Seliem, M.M., Andrisano, V. and El-Maali, N.A. (2004). Osteryoung Square Wave Stripping Voltammetry at Mercury Film Electrode for Monitoring Ultra Levels of Tarabine PFS and its Interaction With SsDNA. J Pharm. Biomed. Anal. 34: 879-890. El-Hafnawey, G.B., El-Hallag, I.S., Ghoneim, E.M. and Ghoniem, M.M. (2004), Voltammetric Behavior and Quantification of the Sedative-hypnotic Drug Chlordiazepoxide in Bulk Form, Pharmaceutical Formulation and Human Serum at a Mercury Electrode. J of Pharm. And Biomed Anal. 34: 75-86.

220 El-Maali, N.A., Osman, A.H., Aly, A.A.A., Al-Hazmi, G.A.A. (2005). Voltammetric Analysis of Cu(II), Cd(II) and Zn(II) Complexes and Their Cyclic Voltammetry with Several Cephalosporin Antibiotics. Bioelectrochemistry. 65: 95-104. Enker, C.G. (2003). The Art and Science of Chemical Analysis. New York: John Wiley and Sons. Ensafi, A.A., Khayamian, T. and Khaloo, S.S. (2004). Application of Adsorptive Cathodic Differential Pulse Stripping Method for Simultaneous Determination of Copper and Molybdenum Using Pyrogallol Red. Anal. Chim. Acta. 505 : 201207. Erdogan, A. (2004). The Aflatoxin Contamination of Some Pepper Types Sold in Turkey. Chemosphere. 56: 321-325. Erk, N. (2004). Voltammetric Behaviour and Determination of Moxifloxacin in Pharmaceutical Products and Human Plasma. Anal. Bioanal.Chem. 378: 13511356. Etzel, R.A. (2002). Mycotoxin. J of Am.Med.Assoc. 287: 425-427. EU Commision, Commision Regulation (EC) No 466/2001 of 8 March 2001 Setting Maximum Levels for Certain Contamination Foodstuffs. (2001). J European

Community. L7.1 Ewing, G.W (Ed). (1997). Analytical Instrumentation Handbook: Revised and

Expanded, 2 nd Ed. New York: Marcel Dekker.

221 Fang, Y., Gong, F., Fang, X. and Fu, C. (1998). Separation and Determination of Cyclodextrins by Capillary Electrophoresis With Aperometric Detection. Anal.

Chim. Acta. 369 : 39-45. Faraj, M.K., Smith, J.E. and Harren, G. (1991). Introduction of Water Activity and Temperature on Aflatoxin Production by Aspergillus flavus and Aspergillus

parasiticus in Irradiated Maize Seeds. Food Additives Contaminants. 6(6): 731736. Farghaly, O.A. and Ghandour, M.A. (2005). Square Wave Stripping Voltammetry for Direct Determination of Eight Heavy Metals in Soil and Indoor-airborne Particulate Matter. Environ. Research. 97: 229-235. Farghaly, O.A., Taher, M.A., Naggar, A.H. and El-Sayed,A.Y., (2005). Square Wave Anodic Stripping Voltammetric Determination of Metoclopramide in Tablets and Urine at Carbon Paste Electrode. J Pharm. Biomed. Anal. 38: 14-20. Farias, P.A.M., Wargener, A.L.R., Bastos, M.B.R., de Silva, A.T. and Castro, A.G.(2004). Cathodic Adsorptive Stripping Voltammetric Behavior of Guanine in the Presence of Copper at the Static Mercury Drop Electrode. Talanta . 61 : 829-835. Feroncova, A., Korgova, E., Miko, R. and Labuda, J., (2001). Determination of Tricyclic Antidepressent Using a Carbon Paste Electrode Modified with βCyclodextrin. J. of Electroanalytical Chem. 492 : 74-77. Ferreira, V.S., Zanoni, M.V.B. and Fogg, A.G. (1999). Cathodic Stripping Voltammetric Determination of Ceftazidine With Reactive Accumulation at a Poly-L-lysine Modified Hanging Mercury Drop Electrode. Anal. Chim. Acta.

384: 159-166.

222 Fifield, F.W. and Kealey, D. (2000). Principle and Practice of Analytical

Chemistry 3 rd Edition. UK: Blackwell science. Fifield, F.W. and Haines, P.J. (2000).Environmental Analytical Chemistry 2 nd Ed, UK: Blackwell Science. Foog, A.G. and Wang, J. (1999).Terminology and Convention for Electrochemical Stripping Analysis. Pure Appl. Chemistry. 71: 891-897. Fraga, J.M.G., Abizanda, A.I.J., Moreno, F.J. and Leon, J.J.A. (1998). Application of Principal Component Regression to the Determination of Captopril by Differential Pulse Polarography With no Prior Removal of Dissolved Oxygen.

Talanta. 46: 73-82 Franco, C.M., Fente, C.A., Vazquez, B.T., Cepede, A., Mahuzier, G. and Prognon, P. (1998). Interaction Between Cyclodextrin and Alatoxins Q1, M1 and P1 Flourescence and Chromatographic Studies. J of Chromatogr. A. 815: 21-29. Fremy, J.M. and Chang, B. (1983). Rapid Determination of AFM1 in Whole Milk by Reversed-phase High Performance Liquid Chromatography. J of

Chromatogr. A. 219: 156-161. Fremy, J.M. and Chu, F.S. (1984). Direct Enzyme-linked Immunosorbent Assay for Determining Aflatoxin M1 at Picogram Levels in Dairy Products. J. Assoc.

Anal. Chem. 67: 1098 – 1101. Fukayama, M. Winterli, W. and Hsieh, D.P.H (1980). Rapid Method for Analysis of Aflatoxin M1 in Dairy Products. J. Assoc. Anal. Chem. 63: 927 – 930.

223 Gao, W., Song, J. and Wu, N. (2004). Voltammetric Behavior and Square-wave Voltammetric Determination of Trepibutone at a Pencil Graphite Electrode. J.

Electroanalytical Chem. 576(1): 1-7. Garcia-Villanova, R.J., Cordon, C., Parama,s A.M.G., Aparicio, P. and Rosales, M.E.G. (2004). Simultaneous Immunoaffinity Column Clean up and HPLC Analysis of Aflatoxins and Ochratoxin A in Spanish Bee Pollen. J Agric. Food

Chem.52: 7235-7239. Garden, S.R. and Strachan, N.J.C. (2001). Novel Colorimetric Immunoassay for the Detection of Aflatoxin B1, Anal. Chim. Acta. 444 : 187-191. Gauch, R., Leuenberger, U. and Baumgartne, E. (1979). Rapid and Simple Determination of Aflatoxin M1 in Milk in the Low Parts per 1012 range. J of

Chromatogr. A. 178: 543-549. Garner, R.C., Whattam, M.M. , Taylor, P.J.L. and Stow, M.W. ( 1993). Analysis of United Kingdom Purchased Spices for Aflatoxins Using Immunoaffinity Column Clean up Procedure Followed up by High-performance Liquid Chromatographic Analysis and Post-column Derivatization with Pyridium Bromide Perbromide. J. Chromatography. 648: 485-490. Gazy, A.A.K. (2004). Determination of Amlodipine Besylate by Adsorptive SquareWave Anodic Stripping Voltammetry on Glassy Carbon Electrode in Tablets and Biological Fluids. Talanta. 62: 575-582. Ghoneim, M.M. Hassanein, A.M., Hamman, E. and Beltagi, A.M. (2000). Simultanoues Determination of Cd, Pb, Cu, Sb, Bi, Se, Zn, Mn, Ni, Cr and Fe in Water Sample by Differential Pulse Stripping Voltammetry at a Hanging Mercury Drop Electrode. Fresenius J. Anal. Chem. 367 : 378-383.

224 Ghoneim, M. M., Tawfik, A. and Khashaba, P.Y. (2003a). Cathodic Adsorptive Stripping Square-wave Voltammetric Determination of Nifedipine Drug in Bulk, Pharmaceutical Formulation and Human Serum. Anal. Bioanal. Chem.

375: 369-375. Ghoneim, M.M., and Tawfik, H.(2004). Assay of Anti-coagulant Drug Warfarin Sodium in Pharmaceutical Formulation and Human Biological Fluids by Square-wave Adsorptive Cathodic Stripping Voltammetry. Anal. Chim. Acta.

511: 63-69. Ghoneim, M.M., El Reis, M.A., Hamman, E. and Beltagi, A.M. (2004). A Validated Stripping Voltammetric Procedure for Quantification of the Anti-hypertensive and Benign Prostatic Hyperplasia Drug Terazosine in Tablets and Human Serum. Talanta. 64(3): 703-710. Ghoneim, M.M., El-Baradie, K.Y. and Tawfik, H. (2003b). Electrochemical Behavior of the Antituberculosis Drug Isonianid and its Square-wave Adsorptive Stripping Voltammetric Estimation in Bulk Form, Tablets and Biological Fluids at a Mercury Electrode. J. Pharm. Biomed. Anal. 33: 673-685. Ghoniem, M.M. and Beltagi, A.M. (2003). Adsorptive Stripping Voltammetric Determination of the Anti-inflammatory Drug Celecoxib in Pharmaceutical Formulation and Human Serum. Talanta, 60: 911-921. Ghoniem, M.M. and Tawfik A. (2004). Assay of Anti-coagulant Drug Warfarin Sodium in Pharmaceutical Formulation and Human Biological Fluids by Square-wave Adsorptive Cathodic Stripping Voltammetry. Anal. Chim. Acta.

511: 63-69.

225 Gimeno, A. and Martins, M. L (1983). Rapid Thin Layer Chromatographic Determination of Patulin, Citrinin and Aflatoxin in Apples and Pears and Their Juices and Jams. J. Assoc. Anal. Chem., 66: 85-91. Goldblatt L.A (1969) Aflatoxin. New York: Academic Press. Gonzalez, M.P.E., Mattusch, J. and Wennrich, R. ( 1998). Stability and Determination of Aflatoxins by High-performance Liquid Chromatography With Amperometric Detection. J.Chromatography A. 828: 439-444. Gonzalez, G., Hinojo, M.J., Mateo, R., Medina, A. and Jimenez, M. (2005). Occurrence of Mycotoxin Producing Fungi in Bee Pollen. Int. J. Food

Microbiology. Article in press. Gooding, J.J., Compton, R.G., Brennan, C.M. and Atherton, J.H. (1997). A New Electrochemical Method for the Investigation of the Aggregation of Dyes in Solution. Electroanalysis. 9(10): 759-764. Gosser, D.K (1993). Cyclic Voltammetry. New York: Wiley-VCH. Goto, T., Matsui, M. and Kitsuwa, T. (1988). Determination of Aflatoxins by Capillary Column gas Chromatography. J of Chromatography. 447: 410-414. Gourama, H. and Bullerman, L. (1995). Aspergillus flavus and Aspergillus

parasiticus: Aflatoxigenic Fungi of Concern in Foods and Feeds: a Review. J of Food Protection. 58: 1395-1404. Gowda, N.K.S., Malathi, V. and Suganthi, R.U. (2004). Effect of Some Chemical and Herbal Compounds on Growth of Aspergillus parasiticus and Aflatoxin Production. Anim. Feed Sci. Technol. 116(3-4): 281- 291.

226 Grigoriadou, I.K., Eleftheriadou, A., Mouratidou, T. and Katikou, P. (2005). Determination of Aflatoxin M1 in Ewe’s Milk Samples and the Produced Curd and Feta Cheese. Food Control. 16 : 257-261. Guiberteau, A., Galcano, T., More, N., Parilla, P. and Salinas, F., (2001). Study and Determination of the Pesticide Imidacloprid by Square-wave Adsorptive Stripping Voltammetry. Talanta. 53: 943-949. Gurbay, A., Aydin, S., Girgin, G., Engin, A.B. and Sahin, G. (2004). Assessment of Aflatoxin M1 Levels in Milk in Ankara, Turkey. Food Control. Article in press. Haghighi, B., Thorpe, C., Pohland, A.E. and Barnette, R. (1981). Development of a Sensitive High performance Liquid Chromatographic Method for Detection of Aflatoxins in Pistachio Nuts. J. of Chromatography. 206: 101-108. Hall, A.J. and Wild, C.P. (1994). The toxicity of aflatoxin: Human, Health,

Veterinary and Agriculture Significance. New York: Academic. Hamilton, F.W. and Ellis, J. (1980). Determination of Arsenic and Antimony in Electrolytic Copper by Anodic Stripping Voltammetry at a Gold Film Electrode.

Anal. Chim. Acta. 119: 225-233. Harvey, D. (2000). Modern Analytical Chemistry. USA: McGraw Hill. Heathcote, J.G. (1984). Aflatoxins and Related Toxins. In: Betina, V. (Ed).

Mycotoxins – Production, Isolation, Separation and Purification. Netherland: Elsevier Science. Heathcote, J.G. and Hibbert, J.R (1984) Aflatoxin: Chemical and Biological Aspect. New York: Elsevier.

227 Heberneh, G.G. (2004). Highlight on Plant Toxin in Toxicon. Toxicon . 44 : 341-344. Hernandez, P., Dabrio-Ramos, M., Paton, F., Ballesteros, Y. and Hernandez, L. (1997). Determination of Abscisic Acid by Cathodic Stripping Square Wave Voltammetry. Talanta. 44: 1783-1792. Herrman, T. (2004). Mycotoxin in feed grains and ingredient in www. oznet.ksu.edu. Quote dated: 18 th April 2004 Heyrovsky, J. and Zuman, P. ( 1968). Practical Polarography; An Intoduction for

Chemistry Students. New York: Academic Press. Hilali, A., Jimenez, J.C., Callejon, M., Bello, M.A. and Guirem, A. (2003). .Electrochemical Reduction of Cefminox at the Mercury Electrode and its Voltammetric Determination in Urine. Talanta. 59(1): 137-146. Hocevar, S.B., Svancara, I., Vytras, K. and Ogorevc, B. (2005). Novel Electrode for Electrochemical Stripping Analysis Based on Carbon Paste Modified With Bismuth Powder. Electrochim. Acta. Article in press. Holcomb, M. , Wilson, D.M., Truckess, M. W. and Thompson, H. C. (1992). Determination of Aflatoxins in Food Products by Chromatography. J. of

Chromatography A. 624: 341-352. Hourch, M.E., Dudoit, A. and Amiard, J-C. (2003). Optimization of New Voltammetric Method for the Determination of Metallothionein.

Electrochim.Acta, 48: 4083-4088. Hussien, H.S. and Brasel, J.M. (2001). Mycotoxin, Metabolism and Impact on Humans and Animals. J Toxicology. 167(2): 101-134.

228 Hutton, E.A., Hocevar, S.B. and Ogorevc, B. (2005). Ex situ Preparation of Bismuth Film Microelectrode for Use in Electrochemical Stripping Microanalysis. Anal.

Chim. Acta. 537 : 285-292. Ibrahim, M.S., Kamal, M.M. and Temerk. Y.M. (2003). Comparison of the Voltammetric Studies at Mercury Electrode and Glassy Carbon Electrodes for the Interaction of Lumichrome with DNA and Analytical Application. Anal.

Bioanal.Chem. 375: 1024-1030. Ibrahim, M.S., Shehatta I.S. and Sultan, M.R. (2002). Cathodic Adsorptive Stripping Voltammetric Determination of Nalidixic Acid in Pharmaceuticals, HumanUrine and Serum. Talanta. 56: 471-579. Illkovic, D. (1934). Polarography Study With the Dropping Mercury Cathode. Part XLIV- The Dependence of Limiting Currents on the Diffusion Constant, on the Rate of Dropping on the Size Electrode. Collection of Czechoslovak chemical

communication. 6 : 498-513. Inam, R. and Somer, G. (1998). Simultaneous Determination of Selenium and Lead in Whole Blood Samples by Differential Pulse Polarography. Talanta. 46: 13471355. International Agency for Research on Cancer (1993). IARC Monographs on the Evaluation of Carcinogenic Risk to Humans, vol. 56. World Health Organisation, Lyon. IUPAC. (1978). Nomenclature: Symbols, Units and Their Usage in Spectrochemical Analysis-II. Spectrochim. Acta, Part B. 32 : 242.

229 Iwamoto, M., Webber, A. and Osteryoung, A. (1984). Cathodic Reduction of Thyroxine and Related Compounds on Silver Electrode. Anal. Chem. 56 : 12031206. Jaimez, J., Fente, C.A., Vazquez, B.I., Franco, C.M., Cepeda, A., Mahuzier, G. and Prognon, P. (2000). Application of the Assay of Aflatoxins by Liquid Chromatography with Fluorescence Detection in Food Analysis. J.

Chromatography A.. 882: 1-10. Janssens, M. M. T., Put, H. M. C. and Nout, M. J. R. (1996). In Vries, Food Safety

and Toxicity. New York: CRC Press. Jeffrey, A.M. and Williams, G.M. (2005). Risk Assessment of DNA-reactive Carcinogens in Food. Toxicology and Appl. Pharmacology. 207(2): 628-635. Jeffery, H.W., Lenorich, L.M and Martin, R. Jr. (1982). Liquid Chromatography Determination of Aflatoxins in Artificially Contaminated Cocoa Beans. J.

Assoc. Off. Anal. Chem. 80 : 1215-1219. Jelen, F., Tomschik, M. and Palecek, E. (1997). Adsorptive Striping Square-wave Voltammetry of DNA. J. Electroanal. Chem. 423 : 141-148. Ji, Z., Yuan, G., Cai, S., Liu, J. and Liu, G. (2004). Electrochemical Studies of the Oxidation Mechanism of Polyamide on Glassy Carbon Electrode, J. of

Electroanalytical Chem. 570: 265-273. Jin, G-P. and Lin, X-Q. (2005). Voltammetric Behavior and Determination of Estrogens at Carbamylcholine Modified Paraffin-impregnated Graphite Electrode. Electrochim. Acta. 50 : 3556-3562.

230 Johnson, W.W., Harris, T.M. and Guyerich V.P. (1996). Kinetics and Mechanism of Hydrolysis of AFB1 exo-8-9-epoxide and Rearrangement of the Dihydrodiol. J.

Am. Chem. Soc. 118: 672-676. Joshua, H. (1993). Determination ofAflatoxin by Reversed-phase High Performance Liquid Chromatography with Post-column in-line Photochemical Derivatization and Fluorescence Detection. J. Chromatography A. 654: 247-254. Jurado-Gonzalez, A. J., Galindo-Riano, M. D. and Garcia-Vargas, M. (2003). Experimental Designs in the Development of a New Method for the Sensitive Determination of Cadmium in Sea Water by Adsorptive Cathodic Stripping Voltammetry. Anal. Chim. Acta. 487 : 229-241. Kamal, M.M., Abu Zuhri, A.Z. and Nasser, A.A. O. (1996). Adsorptive Stripping Voltammetric Analysis of 2,4,6-tri(2 ’-pyridyl)-1,3,5-triazine at a Mercury Electrode. Fresenius J Anal Chem. 356: 500-503. Kamimura, H., Nishijima, M., Yasuda, K., Ushiyama, H., Tabata, S., Matsumoto, S. and Nishima, T. (1985). Simple, Rapid Clean-up Method for Analysis of Aflatoxins and Comparison with Various Methods. J. Assoc. Off. Anal. Chem.

68: 458-461. Kamkar A. (2004) A Study on the Occurence of Aflatoxin M1 in Raw Milk Produced in Sarab City of Iran. Food Control. 16(7): 593-599. Kamkar, A. (2005). A Study of the Occurrence of Aflatoxin M1 in Iranian Feta Cheese. Food Control. Article in press. Karadjova, I., Girousi, S. , Illadou, E. and Stratis, I. (2000). Determination of Cd, Co, Cr, Cu, Fe, Ni and Pb in Milk, Cheese and Chocolate. Mikrochim. Acta.

134: 185-191.

231 Kefala, G., Economou, A., Voulgaropoulos, A. and Sofoniou, M.(2003). A Study of Bismuth-film Electrode for the Detection of Trace Metals by Anodic Stripping Voltammetry and Their Application to the Determination of Pb and Zn in Tapwater and Human Hair. Talanta. 61: 603-610. Khodari, M., Ghandour, M. and Taha, A.M. (1997). Cathodic Stripping Voltammetric of the Anticancer Agent 5-fluorouracil and Determination in Urine. Talanta. 44: 305-310. Kim, H. J., Chang, S-C. and Shim, Y-B., (2002). α-cyclodextrin Modified Screen Printed Graphite Electrodes for Detection of Phenols. Bull, Korean Chem. Soc.

23: 427-431. Kizek, R., Masarik, M., Kramer, K.J., Potesil, D., Bailey, M., Howard, J.A., Klejdus, B., Mikelova, R., Adam, V., Trimkova, L. and Jelan, F. (2005). An Analysis of Avidin, Biotin and Their Interaction at Attomole Levels by Voltammetric and Chromatographic Techniques. Anal. Bioanal. Chem. 381 : 1167-1178. Koehler, P.E., Benchat, L.R. and Chhinnan, M.S. (1985). Influence of Temparature and Water Activity on Aflatoxin Production by Aspergillus flavus in Cowpea (Vigna Ungiculata) Seeds and Meal. J of Food Protection. 48: 1040-1043. Kok, W. T., Van Neer, T.C.H., Traag, W.A. and Tuinstra, L.G.M.T. (1986). Determination of Aflatoxins in Cattle Feed by Liquid Chromtography and Postcolumn Derivatization with Electrochemically Generated Bromine. J.

Chromatogr. 367: 231 – 236. Kok, W.Th. (1994). Derivatization Reactions for the Determination of Aflatoxins by Liquid Chromatography With Fluorescence Detection. J Chromatography B

659: 127 – 137.

232 Komorsky-Lovric, S., Lovric, M. and Branica, M. (1992). Peak Current–frequency Relationship in Adsorptive Stripping Square-wave Voltammetry. J. Electroanal.

Chem. 335(1-2): 297-308. Kontoyannis, C. G., Antimisianis, S.G. and Douromis, D. (1999). Simultaneous Quantitative Determination of Diazepan and Liposomes using Differential Pulse Polarography. Anal. Chim. Acta. 391: 83-88. Kotoucek, M. Skopilova, J. and Michalkova, D. (1997). Electroanalytical Study of Salozosulfapyridine and Biseptol Components at the Mercury Electrode. Anal.

Chim.Acta. 353: 61-69. Kubiak, W.W., Latonen, R.M., and Ivaska, A. (2001). The Sequential Injection System With Adsorptive Stripping Voltammetry Detection. Talanta. 53: 12111219. Kussak, A., Anderson, B. and Anderson, K. (1995a). Determination of Aflatoxins in Airborne Dust From Feed Factories by Automated Immunoaffinity Column Clean-up and Liquid Chromatography. J Of Chromatogr. A. 708: 55-60. Kussak, A., Anderson, B. and Anderson, K. (1995b). Immunity Column Clean-up for the High Performance Liquid Chromatographic Determination of Aflatoxins B1, B2, G1, G2, M1 and Q1 in Urine. J Of Chromatogr. B. 672: 253-259 Kutner, W., Wang, J., L’Her, M. and Buck, R. (1998). Analytical Aspects of Chemically Modified Electrode: Classification, CriticalEvaluation and Recommendation. (IUPAC Recommendation 1998). Pure and Appl. Chem. 70: 1301-1318. Lafleur, R.D., Myland, J.C. and Oldham, K.B. (1990). Analytical Microelectrode Voltammetry with Minimal Instrumentation. Electroanalysis. 2: 223-238.

233 Lafont, P. and Siriwardana, M. C. (1981). Assay Method for Aflatoxin in Milk. J of

Chromatogr. 219: 162-166. Lam, G.K., Kalvoda, R. and Kaponica, M. (1983). Determination of Uranium by Adsorption Stripping Voltammetry. Anal. Chim. Acta . 154: 79-83. Lau, P.H. and Chu, F.S. (1983). Preparation and Characterisation of Acid Dehydration Product of Aflatoxicol. J. Assoc. Off. Anal Chem. 66(1): 98-101.. Lee, L.S., Wall, J.H., Cotty, P.J. and Bayman, P. (1990). Interpretation of Enzymelinked Immunosorbent Assay with Conventional Chromatography Procedures for Quantitation of Aflatoxin in Individual Cotton Bolls, Seeds and Seeds Sections. J. Assoc. Anal. Chem. 73(4): 581-584. Leontopoulos, D., Siafaka, A. and Markaki, P. (2003). Black Olives as Substrate for

Aspergillus parasiticus Growth and Aflatoxin B1 Production. Food Microbiology. 20: 119-126. Leszezynska, J. Kucharska, U. and Zegota, H. (2000). Aflatoxin in Nuts Assayed by Immunological Methods. Eur Food Res Technol. 210 : 213-215. Levi, C. (1980). Mycotoxins in Coffee. J Assoc. Off. Anal. Chem. 63(6): 12821285. Lin, L., Lawrence, N.S., Thongngamdee, S., Wang, J. and Lin, Y. (2005). Catalytic Adsorptive Stripping Determination of Trace Chromium (VI) at the Bismuth Film Electrode. Talanta. 65: 144-148. Lin, L., Thongngamdee S., Wang, J., Lin, Y., Sadik, O.A. and Ly, S-Y. (2005). Adsorptive Stripping Voltammetric Measurements of Trace Uranium at the Bismuth Film Electrode. Anal. Chim. Acta . 535: 9-13.

234 Lin, L., Zhang, J., Wang, P., Wang, Y. and Chen, J. (1998). Thin-layer Chromatography of Mycotoxins and Comparison with Other Chromatographic Methods. J of Chromatogr. A. 815: 3-20. Liu, J.H., Lin, W.F. and Nicol, J.D. (1980). The application of Anodic Stripping Voltammetry to Forensic Science. II. Anodic Stripping Voltammetric Analysis of Gunshot Residues. Forensic Sci Int. 16: 53-62. Liu, Y., Jiang, Y., Song, W., Lu, N., Zou, M., Xu, H. and Yu, Z. (2000). Voltammetric Determination of 5-hydroxydole-3-acetic Acid in Human Gastric Juice. Talanta . 50 : 1261-1266. Locatelli, C. (2005). Overlapping Voltammetric Peaks – an Analytical Procedure for Simultaneous Determination of Trace Metals. Application to Food and Environmental Matrices. Anal Bioanal Chem. 381: 1073-1081. Loftsson, T. and Brewster, M.E. (1996) Pharmaceutical Applications of Cyclodextrins. 1. Drug Solubilisation and Stabilisation. J. Pharm. Sci. 85: 10171025. Lopez, C.E, Ramos, L.L., Ramadan, S.S. and Bulacio, L.C. (2002). Presence of Aflatoxin M1 in Milk for Human Consumption in Argentina. Food Control.

14(1): 31-34. Lupu, A., Compagnone, D. and Palleschi, A. (2004). Screen-printed Enzyme Electrodes for the Detection of Marker Analytes During Wine Making. Anal.

Chim. Acta. 513 : 67-72. Ly, S-Y, Kim,D-H. and Kim, M-H. (2002). Square-wave Cathodic Stripping Voltammetry Analysis of RDX Using Mercury-film Plated Glassy Carbon Electrode. Talanta. 58: 919-926.

235 Malaysian Food Act (1983) Act No 281, Reviewed 2002, Food Quality Control Dept, Ministry of Health, Malaysia. Mackson, J., Wright, G., Rachaputi, N.C., Krosch, S. and Tatnell, J. (1999). Aflatoxin Research Update Workshop Proceeding. Kingaroy, Queensland. August 1999. Martins, M.L. and Martin, H.M.,(2004). Aflatoxins M1 in Yoghurts in Portugal. Int.

J of Food Microbiology. 91: 315-317. Manetta, A.C., Giuseppe, L.D., Giammarco, M.,Fusaro, I., Simonella, A., Gramenzi, A. and Formigoni, A. (2005). High-performance Liquid Chromatography With Post-column Derivatisation and Fluorescence Detection for Sensitive Determination of Aflatoxin M1 in Milk and Cheese. J Chrom. A.

1083 : 219-222. Mendez-Albores J.A., Arambula-Villa G., Preciado-Ortiz R.E. and MorenoMartinez E., (2004). Aflatoxins in Pozol, a Nixtamalized Maize-based Food. Int

J of Food Microbiology. 94 : 211-215. Metrohm (2005). Introduction to Polarography and Voltammetry Monograph, Metrohm Ltd, Switzerland. Micheli, L. Grecco, R., Moscone, D. and Palleschi, G. (2005). An Electrochemical Immunosensor for Aflatoxin M1 Determination in Milk Using Screen-printed Electrodes. Biosensors and Bioelectronic. 21(4): 588-596. Miller, N. (1987). Toxicology Aspects of Food. New York: Elsevier Science. Miller, J.C. and Miller, J.N. (1993). Statistic for Analytical Chemistry. London: Ellis Horwood.

236 Miller, N., Pretorius,H.E. and Trinder, D.W. (1985). Determination of Aflatoxins in Vegetable Oils. J. Assoc. Off. Anal. Chem. 68: 136-137. Mohd. Yusoff, A.R., Ahamad, R. and Azman, S. (1998). Kaedah Voltammetri Dalam Penentuan Pewarna reaktif dan Hasil Hidrolisisnya. Malays. J. Anal. Sci.

4(1): 139-146. Molina, M. and Gianuzzi, L. (2002). Modelling of Aflatoxin Production by

Aspergillus parasticus in a Solid Medium at Different Temperature, pH and Propionic Acid Concentration. Food Res. Int. 35: 585-594. Moressi, M.B., Andreu, R., Calvente, J.J, Fernandez, H. and Zon, M. A. (2004). Improvement of Alterneriol Monomethyl Ether Detection at Gold Electrode Modified With a Dedocanethiol Self-assembled Monolayer. J. of

Electroanalytical Chem. 570: 209-217. Morfobos, M., Economou, A. and Voulgaropoulos, A. (2004). Simultaneous Determination of Nickel(II) and cobalt(II) by Square Wave Adsorptive Stripping Voltammetry on a Rotating-disc Bismuth-film Electrode. Anal Chim Acta. 519: 57-64. Mouley, Y. (1984). Biochemical Effects of Mycotoxin. In Betina, V (Ed.).

Mycotoxin – Producton, Isolation, Separation and Purification. Netherland: Elsevier Science. Nawaz, S., Coker, R.D and Haswell, S.J. (1992). Development and Evaluation of Analytical Methodology for Determination of Aflatoxins in Palm Kernel.

Analyst. 117 : 67 – 71.

237 Navalion, A., Blanc, R., Reyes, L., Navas, N. and Vikches, J.K. (2002). Determination of the Antibacterial Enrofloxacin by Differential Pulse Adsorptive Stripping Voltammetry. Anal. Chim. Acta . 454: 83-91. Neal, G.E. (1987). Influence of Metabolism: AflatoxinMetabolism and its Possible Relationships With Disease. In: Watson D.H. Natural Toxicants in Foods:

Progress and prospects. London: VCH. Niedwetzki, G. Lach, G. and Ceschwill, K. (1994). Determination of Aflatoxin in Food by Use of an Automatic Work Statio. .J.Of Chromatography A. 661: 175180. Nilufer, D. and Boyacio, D. (2002). Comparative Study of Three Different Methods for the Determination of Aflatoxins in Tahini. J. Agric. Food. Chem. 50: 33753379. Nouws, H.P.A., Delerue-Matos, C., Barros, A.A. and Rodrigues, J.A., (2005a). Electroanalytical Study of the Antidepressant Sertraline. J. Pharm. Biomed.

Anal. 39(1-2): 290-293. Nouws, H.P.A., Delerue-Matos, C., Barros, A.A., Rodrigues, J.A. and Silva, A.S. (2005b). Electroanalytical Study of Fluvoxamine. Anal and Bioanal. Chem.

382(7): 1662-1668. Official Methods of Analysis 14 th Ed. (1984) AOAC, Washington D.C. Ohfuji, K., Sato, N., Hamadansoto, N., Kabayashi, T., Imada, N., Okura, H. and Watanabe, E., (2004). Construction of a Glucose Based on a Screen-printed Electrode and a Novel Mediator Pyocyanin From Pseudomonos Aeruginosa.

Biosensors and Bioelectronic. 19: 1237-1244.

238 Orinakova, R., Tankova, L., Galova, M. and Supicova, A. (2004). Application of Elimination Voltammetry in the Study of Electroplating Processes on the Graphite Electrode. Electrochimica Acta. 49 : 3587-3594. Ortiz, M.Z., Arcos, J., Juarros, J.V., Lopez-Palacios, J. and Sarabia, L.A. (1993). Robust Procedure for Calibration and Calculation of the Detection Limit of Trimipramine by Adsorptive Stripping Voltammetry at a Carbon Paste Electrode. Anal. Chem. 65: 678-682. Ozkan, S.A. and Uslu, B. (2002). Electrochemical Study of Fluvastatin Sodium – Analytical Application to Pharmaceutical Dosage Forms, Human Serum and Simulated Gastric Juice. Anal. Bioanal.Chem. 372: 582-586. Ozkan, S.A., Uslu, B., Aboul-Enein, H.Y. (2003). Voltammetric Investigation of Tamsulosin. Talanta. 61: 147-156. Pacheco, W.F., Farias, P.A.M. and Aucelio, R.Q. (2005). Square-wave Adsorptive Stripping Voltammetry for the Determination of Cyclofenil After Photochemical Derivatisation. Anal. Chim. Acta. 549(1-2): 67-73. Padano, M. L. and Rivas, G. A., (2000). Amperometric Biosensor for the Quantification of Gentisic Acid using Poliphenol Oxidase Modified Carbon Paste Electrode. Talanta. 53: 489-495. Papp E., Otta K.H., Zaray G. and Mincsvics (2002). Liquid Chromatographic Determination of Aflatoxins. Microchemical J . 73(1-2): 39-46. Pare, J.R.J and Belanger, J.M.R. (1997). Instrumental Methods in Food Analysis. N.York : Elsevier.

239 Parham, H and Zargar, B. (2001). Determination of Isosorbic Dinitrate in Arterial Plasma, Synthetic Serum and Pharmaceutical Formulations by Linear Sweep Voltammetry on a Gold Electrode. Talanta. 55: 255-262. Park, K.Y. and Bullerman. L.B. (1983). Effects of Cycling Temperature on Aflatoxins Production by Aspergillus parasiticus and Aspergillus flavous in Rice and Cheese. J of Food Science. 48: 889-896. Park, D.L. and Rua, S.M. (1991). Sample Collection and Preparation Techniques for Aflatoxin Determination in Whole Cottonseed. J Assoc. Off. Anal. Chem. 74 : 73-75. Parlim, A. and Zerger, S. (2003). Determination of Isosorbide Synthetic Serum and Pharmaceutical Formulation by Linear Sweep Voltammetry on a Gold Electrode. Talanta. 55: 255-262. Patey A.L., Sharman M. and Gilbert J (1990). Determination of Aflatoxin B1 Levels in Peanut Butter Using an Immunoaffinity Column Cleanup Procedure: Interlaboratory Study. Food Addit. Contam. 7; 515-520. Patey, A.L., Sharman, M. and Gilbert, J. (1991). Liquid Chromatographic Determination of Aflatoxin Levels in Peanut Butters Using an Immunoaffinity Column Clean up Method: International Collaborative Trial. J. Assoc. Off. Anal.

Chem. 74: 76-81. Pavlova V., Mirceski V., Lovric S.K, Javanovic S.P. and Mitrevski B. (2004). Studying Electrode Mechanism and Analytical Determination of Cocaine and its Metabolites at the Mercury Electrode Using Square-wave Voltammetry. Anal

Chim.Acta . 512: 49-56.

240 Pena, R. Alcaraz, M.C., Arce, L., Rios, A. and Valcarcel, M. (2002). Screening of Aflatoxins in Feed Samples Using a Flow System Coupled to Capillary Electrophoresis. J Of Chromatography A. 967: 303-314. Perez-Ruiz, T., Martinez-Lozano, C., Sanz, A. and Guillen, A. (2004). Successive Determination of Thiamine and Ascorbic Acid in Pharmaceuticals by Flow Injection Analysis. J.Pharm. Biomed. Anal. 34: 551-557. Pesavento, M., Domagala, S., Baldini, E. and Cucca, L. (1997). Characterisation of Enzyme Linked Immunosorbent Assay for Aflatoxin B1 Based on Commercial Reagents. Talanta. 45: 91-104. Pestka, J. and Bundy, G. (1990). Alteration of Immune Function Following Dietary Mycotoxin Exposures. Canadian J. of Physilogy and Pharmacology. 68 : 10091016. Pezzatini, G., Midili, I., Toti, G., Loglio, F. and Innoceti, M. (2004). Determination of Chlorite in Drinking Water by Differential Pulse Voltammetry on Graphite.

Anal. Bioanal.Chem. 380 : 650 - 657. Pildain, M.B., Vaamonde, G. and Cabral, D. (2004). Analysis of Population Structure of Aspergillus flavus From Peanut Based Vegetative Compatibility, Geographic Origin, Mycotoxin and Sclerotia Production. Int. J. Food Microb.

93: 31- 40. Plattner, R.D., Bennett, G.A. and Stubblefield, R.D. (1984). Identification of Aflatoxins by Quadrupole Mass Spectrometry/Mass Spectrometry. J. Assoc.

Anal. Chem. 67(4): 734 -738.

241 Pons, W.A. Jr. and Franz, A.O. Jr. (1977). High Performance Liquid Chromatography of Aflatoxins in Cottonseed Products. J Assoc off Anal Chem.

60: 89 - 95. Pournaghi-Azar, M.H. and Dastango, H. (2002). Differential Pulse Anodic Stripping Voltammetry of Copper in Dichloromethane: Application to the Analysis of Human Hair. Anal. Chim. Acta. 405:135 - 144. Rachaputi, N.C., Wright, G.C., Krosch, S. and Tatnell,J. (2001). Management Practice to Reduce Aflatoxin Contamination in Peanut. Proceedings of the 10th Australian Agronomy Conference, Hobart. 2001. Radi, A. (2003). Square-wave Adsorptive Cathodic Stripping Voltammetry of Pantoprazole. J of Pharmaceutical and biomedical analysis. 33: 687 – 692. Radi, A. and El-Sherif, Z. (2002). Determination of Levofloxacin in Human Urine by Adsorptive Square-wave Anodic Stripping Voltammetry on a Glassy Carbon Electrode. Talanta. 58: 319 - 324. Radi, A., Beltagi, A.M. and Ghoniem, M.M. (2001). Determination of Ketorolac in Human Serum by Square-wave Adsorption Stripping Voltammetry. Talanta. 54 : 283 - 289. Rasooli, I. and Abnayeh, M.R.(2004). Inhibitory Effect of Thyme Oils on Growth and Aflatoxin Production by Aspergillus parasiticus. Food Control. 15: 479 483. Rastogi, S., Das, M. and Khanna, S.K. (2001). Quantitative Determination of Aflatoxin B1-oxime by Column Liquid Chromatography With Utraviolet Detection. J Of Chromatography A. 933: 91 - 97.

242 Rastogi, S., Dwivedi, P.D., Khanna, S.K. and Das, M. (2004). Detection of Aflatoxin M1 Contamination in Milk and Infant milk Products From Indian Market by ELISA. Food Control. 15 : 287 - 290. Reif, K. and Metzger, W. (1995). Determination of Aflatoxins in Medicinal Herbs and Plant Extracts. J Of Chromatography A. 692: 131 - 136. Reinke, R. and Simon, J. (2002). The Online Removal of Dissolved Oxygen From Aqueous Solutions Used in Voltammetric Technique by Chromatomembrane Method. Anal. Bioanal. Chem. 374 : 1256 - 1260. Riley, T. and Tomlinson, C. (1984). Principles of Electroanalytical Methods

(Analytical Chemistry by Open Learning). London: John Wiley. Risk assesment studies report No. 5 (1991). Chemical Hazrad – Evaluation Aflatoxin in Food. Food and Environmental Hygeine Department, Hong Kong. Rizk, M., Belal, F., Aly, F.A. and El-Enany, N.M. (1998). Differential Pulse Polarographic Determination of Ofloxacin Pharmaceuticals and Biological Fluids. Talanta. 46: 83 - 89. Rizzo I., Vedoya G., Maurutto S., Haidukowski M. and Varsavsky E., (2004). Assessment of Toxigenic Fungi on Argentinean Medicinal herbs.

Microbiological Research. 159 : 113 - 120. Rodriguez, J., Berzas, J.J., Castanada, G. and Rodrigues, N., (2005). Voltammetric Determination of Imanitib (Gleevec) and its Main Metabolite Using Squarewave and Adsorptive Stripping Square-wave Technique in Urine Samples.

Talanta. 66: 202 - 209.

243 Rodriguez, J., Berzas, J.J., Castanada, G. and Rodrigues, R. (2004). Determination of Seldenafil Citrate (Viagra) and its Metabolite (UK-103,320) by Square-wave and Adsorption Stripping Square-wave Voltammetry. Total Determination in Biological samples. Talanta. 62: 427 - 432. Sabry, S.M. and Wahbi, A.A.M. (1999). Application of Orthogonal Functions to Differential Pulse Voltammetric Analysis: Simultanuoes Determination of tin and Lead on Soft Drinks. Anal. Chim. Acta, 401: 173 - 183. Safavi, A., Maleki, N. and Shahbaazi, H.R.(2004). Indirect Determination of Cyanide Ion and Hydrogen Cyanide by Adsorptive Stripping Voltammetry at a Mercury Electrode. Anal.Chim.Acta. 503: 213 - 221. Salavagione. H.J., Arias. J., Garces. P., Marallon. E., Barbero. C. and Vazguez. J.L, (2004). Spectroelectrochemical Study of the Oxidation of Aminophenols on Platinum Electrode in Acid Medium. J. Of Electroanalytical Chem. 565: 375 383. Saleemullah, Iqbal, A., Khalil, I.A. and Shah H. (2005). Aflatoxin Contents of Stored andArtificially Inoculated Cereals and Nuts. Food Chemistry. Article in press. Salleh, B. (1998). Mikotoksin- Implikasi Terhadap Kesihatan Manusia dan Haiwan. Pulau Pinang: Universiti Sains Malaysia. Samajpathi, N. (1979) .Aflatoxin Produced by Five Species of Aspergillus on Rice.

Naturwissenschaften. 66 : 365 - 366. Sandulescu, R., Mirel, S. and Oprean, R. (2000). The Development of Spectrophotometric and Electroanalytical Methods for Ascorbic Acid and

244 Acetaminophen and Their Application in the Analysis of Effervescent Dosage Form. J. Pharm. Biomed. Anal. 23: 77 - 87. Sanna, G., Pilo, M.I., Pin, P.C. Tapparo, A. and Seeber, R. (2000). Determination of Heavy Metals in Honey by Anodic Stripping Voltammetry at Microelectrode.

Anal. Chim. Acta. 415: 165 - 173. Santos, M.C. and Machado, S.A.S. (2004). Microgravimetric, Rotating Ring-disc and Voltammetric Studies of the Underpotential Deposition of Selenium on Polycrystalline Platinum Electrode. J. of Electroanalytical Chem. 567: 203 210. Sarimehmetoglu, B., Kuplulu, O. and Celik, T.H. (2004). Detection of Aflatoxin M1 in CheeseSamples by ELISA. Food Control. 15 : 45 - 49. Sassahara, M., Netto, D.P. and Yanaka, E.K. (2005). Aflatoxin Occurence in Foodstuff Supplied to Dairy Cattle and Aflatoxin M1 in Raw Milk in the North of Parana State. Food and Chemical Technology. 43: 981 - 984. Scholten, J.M and Spanjer, M.C. (1996). Determination of Aflatoxin B1 in Pastachio Kernels and Shells. J. Assoc. Off. Anal. Chem. 79: 1360 - 1364. Scott, P.M. and Lawrence, G.A. (1997). Determination of Aflatoxins in Beer. J.

Assoc. Off. Anal. Chem. 80 : 1229 - 1233. Setamou, M.K., Gardwell, F., Schulthess, S. and Hell, K. (1997). Aspergillus flavus Infection and Aflatoxin Contaminants of Preharvest Maize in Benin. Plant

Disease. 81: 1327 - 1327. Shahrokhian, S. and Karimi, M. (2004). Voltammetric Studies of a cobalt(II)-4Methylsalophen Modified Carbon-paste Electrode and its Application for the

245 Simultaneous Determination of Cysteine and Ascorbic Acid. Electrochim. Acta.

50: 77 - 84. Shams, E., Barbei, A. and Soltaninezhad,M. (2004). Simultaneous Determination of Copper, Zinc and Lead by Adsorptive Stripping Voltammetry in the Presence of Morin. Anal. Chim. Acta. 501: 119 - 124. Shannon, G.M., Shotwell, O.L and Kwole, W.F. (1983). Extraction and Thin Layer Chromatography of Aflatoxin B1 in Mixed Feeds. J. Assoc. Off. Anal. Chem. 66: 582 - 586 Shenasi, M., Aidoo, K.E. and Candlish, A.A.G. (2002). Microflora of Date Fruits and Production of Aflatoxins at Various Stages of Maturation. Int.J Food

Microbiology. 79(1-2): 113 - 119. Shi K. and Shiu K.K. (2004). Adsorption of Some Quinone Derivatives at Chemically Activated Glassy Carbon Electrode. J. of Electroanalytical Chem.

574(1): 63 - 70. Shotwell, O.L and Goulden, M.L (1977). Aflatoxins: Comparison of Analysis of Corn by Various Methods. J. Assoc. Off. Anal. Chem. 60 : 83 - 88. Shotwell, O.L, Goulden, M.L., Benntt, G.A., Plattner, R.D. and Hesseltine, C.W (1977). Survey of 1975 Wheat and Soybeans for Aflatoxins, Zeralenone and Ochratonin, J. Assoc. Off. Anal. Chem. 60: 778 - 783. Shotwell, O.L., Hesseltin,e C. and Stubblefield, R.D. (1966). Production of aflatoxin on rice. Appl. Microbiol. 14: 425 - 428.

246 Simon, P., Delsaut, P., Lafontaine, M. Morele, Y. and Nicol, T. (1998). Automated Column-switching High-performance liquid chromatography for the determination of aflatoxin M1. J of Chromatogr. B. (1998). 712: 95 - 104. Skoog, D.A., West, D.M. and James, F. (1996) Fundamentals of Analytical

Chemistry. 7th Ed. New York; Saunders College Publishing, Skrzypek S., Ciesielski W., Sokolowski A., Yilmaz S. and Kazmierczak A. (2005). Square Wave Adsorptive Stripping Voltammetric Determination of Famotidine in Urine. Talanta. 66:1146 - 1151. Smith, J.E. and Moss M.O. (1985). Mycotoxins – Formation, Analysis and

Significance. Chichester: John Wiley. Smyth, M. R., Lawellin, D. W. and Osteryoung, J.G (1979) Polarographic Study of Aflatoxin B1, B2, G1 and G2: Application of Differential-pulse polarography to the determination of Aflatoxin B1 in Various Foodstuff. Analyst. 104: 73 - 78. Smyth, M.R. and Vos, J.G., (1992). In Shevla, G. (Ed). Analytical Voltammetry in

Comprehensive Analytical Chemistry. Amsterdam: Elsevier Science. Smyth, M.R. and Smyth, W.F. (1978). Voltammetric Methods for the Determination of Foreign Organic Compounds of Biological Significance. A review. Analyst.

103(1227): 103 - 122. Sonthalia, P., McGaw, E., Show, Y. and Swain, G.M. (2004). Metal Ion Analysis in Contaminated Water Samples Using Anodic Stripping Voltammetry and a Nanocrystalline Diamond Thin-film Electrode. Anal Chim Acta. 522 : 35 - 44.

247 Sorensen, L.K. and Elbaek, T. (2005). Determination of Mycotoxins Bovine Milk by Liquid Chromatography Tandem Mass Spectrometry. J Chrom. B. 820 :183196. Stack, M. E. and Pohland, A.E. (1975). Collabration Study of a Method for Chemical Confirmation of the Identify of Aflatoxin. J. Assoc.Off. Anal.Chem.

58: 110 - 113. Stroka, J. and Anklem, E (2002). New Strategies for the Screening and Determination of Aflatoxins and Detection of Aflatoxin-producing Moulds in Food and Feed. Trend In Anal. Chem. 21: 90 - 95. Stroka, J., Elke, A., Urban, J., John, G., Anna, B., Carlo, B., Per-Erik, C., Fiona, G., John, G., and Loius, S. (2000). Immunoaffinity Column Cleanup With Liquid Chromatograhpy Using Post-column Bromination for Determination of Aflatoxins in Peanut Butter, Pistachio Paste, Fig Paste and Paprika Powder. Collaborative Study. J. of AOAC International. 83: 320 - 340. Stubblefield, R.D and Showell, O.L (1977). Reverse Phase Analytical and Preparative High Performance Liquid Chromatography of Aflatoxins. J. Assoc.

Off. Anal. Chem. 60 : 784 - 790. Stubblefield, R.D., Kwolek, W.F. and Stoloff, L (1982). Determination and Thin Layer Chromatographic Confirmation of Identity of AFB1 and AFM1 in Artificially Contaminated Beef Livers: Collaborative Study. J. Assoc. Off. Anal.

Chem. 65: 1435 - 14. Sturm, J.C., Canelo, H., Nunez-Vergara, L.J. and Squella, J.A. (1997). Voltammetric Study of Ketorolac and its Differential Pulse Polarographic Determination in Pharmaceuticals. Talanta. 44: 931 - 937.

248 Sun, N., Mo, W-M. Shen, Z-L and Hu, B-X. (2005). Adsorptive Stripping Voltammetric Technique for the Rapid Determination of Tobramycin on the Hanging Mercury Electrode. J. Pharm. Biomed. Anal. 38(2): 256 - 262. Sun, W. and Jiao, K. (2002). Linear Sweep Voltammetric Determination of Protein Based on its Interaction With Alizarin Red S. Talant. 56: 1073 - 1080. Taguchi, S., Fukushima, S., Summoto, T., Yoshida, S. and Nishimune, T. (1995). Aflatoxins in Foods Collected in Osaka, Japan. J AOAC Int. 78: 325 – 327. Takahashi, D.M. (1977a). Reverse Phase High Pressure Liquid Chromatographic Analytical System for Aflatoxins in Wine With Fluorescence Detection. J

Chromatograph. 131: 147 - 156. Takahashi, D.M. (1977b). Reverse Phase High Pressure Liquid Chromatographic Analytical System for Aflatoxins in Wine and Fruit Juices. J. Assoc. Off. Anal.

Chem. 60: 799 – 804. Tarin, A, Russell, M.G. and Guardino, X. (2004). Use of High Performance Liquid Chromatography in Assess Airborne Mycotoxins. J of Chromatogr. A. 1047(2): 235 - 240. Thompson, M. (1998). Do We Really Need Detection Limit?. Analyst. 123: 405 407. Torriero, A.A.J., Luco, J.M., Sereno, L. and Raba, J. (2004). Voltammetric Determination of Salicylic Acid in Pharmaceuticals Formulations of Acetylsalicylic Acid. Talanta. 62: 247- 254.

249 Tozzi, C., Anfossi, L. Baggiani, C., Giavanalli, C. and Girandi, G. (2003). A Combinatorial Approach to Obtain Affinity Media With Binding Properties Towards the Aflatoxins. Anal. Bioanal. Chem. 375: 994 - 999. Trucksess, M. W. and Stollof, L. (1980). Thin Layer Chromatographic Determination of Alatoxins in Dry Ginger Root and Ginger Oleoresin. J. Assoc.

Off. Anal. Chem.63: 1052 - 1054. Trucksess, M. W., Stack, M. E., Nesheim, S., Page, S.W. , Albert, R.H., Hansen, T.J. and Donahue, K.F. (1991). Immunoaffinity Column Coupled with Solution Fluometry or Liquid Chromatography Postcolumn Derivatization for Determination of Aflatoxins in Corn, Peanuts and Peanut butter: Collaborative Study. J. Assoc. Off. Anal. Chem. 74 : 81 - 88. Trucksess, M. W., Stollof, L., Pons, W.A., Cucullu, A.F. Lee, L.S. and Franz, A.O. (1977). Thin Layer Chromatography of AFB1 in Egg. J. Assoc. Off. Anal. Chem.

60: 795 - 798. Tuinstra L.G.M. and Hassnott, W. (1983). Rapid Determination of Aflatoxin B1 in Dutch Feedingstuffs by High Performance Liquid Chromatography and Postcolumn Derivatisation. J of Chromatogr. 282: 457 - 462. Tuinstra, L.G.M., Kienhuis, P.G.M. and Dols, P. (1990). Automated Liquid Chromatographic Determination of Aflatoxin M1 in Milk Using on-line Dialysis for Sample Preparation. J. Assoc. Off. Anal. Chem. 73: 969 - 973. Turner, P.C., Sylla, A., Gong, Y.Y., Diallo, M.S., Surcliffer, A.E., Hall, A. J. and Wild, C.P. (2005). Reduction in Exposure to Carcinogenic Aflatoxins by Postharvest Intervention Measures in West Africa: Community Based Intervention Study. Lancet. 365: 1950 - 1956.

250 Tutour, L.B., Elaraki, A. T. and Aboussalim, A.(1984). Simultaneous Thin Layer Chromatographic Determination of Aflatoxin B1 and Ochratoxin A in Black Olives, J Assoc Off Anal Chem. 67 : 611 - 620. Urano, T., Trucksess ,M.W. and Page, S.W., (1993). Automated Affinity Liquid Chromatography Sstem for On-line Isolation, Separation and Quantitation of Aflatoxins in Methanol-water Extracts of Corn or Peanuts. J.Agric. Food Chem.

41: 1982 - 1985. Vahl, M. and Jorgensen, F. (1998). Determination of Aflatoxins in Food Using LC/MS/MS. Z Lebensm Unters Forsch A.. 206: 243 - 245. Van Egmond, H.P. (1989). Introduction of Mycotoxins in Dairy Products. London: Elsevier Applied Science. Verma, K.K., Jain, A., Verma, a. and Chaurasia, A. (1991). Spectrophotometric Determination of Ascorbic Acid in Pharmaceuticals by Background Correction and Flow Injection. Analyst. 116: 641 - 645. Versantroort, C.H.M., Oomen, A.G., and Sips, A.J.A.M. (2005). Applicability of an in Vitro Digestion Model in Assessing the Bioaccessibility of Mycotoxins From Food. Food and Chemical Toxicology. 43 : 31- 40. Volke, J. and Liska, F. (1994). Electrochemistry in Organic Synthesis. New York: Springer-Verlag. Volkov, A.G. and Mwesigwa, S. (2001). Electrochemistry of Soyabean: Effect of Uncouplers, Pollutant and Pesticides. J. Of Electroanalytical Chemistry. 496: 153 - 157.

251 Wang, H. S., Huang, D. H. and Liu, R.M. (2004). Study on the Electrochemical Behavior of Epinephrine at a Poly(3-methylthiophene) Modified Glassy Carbon Electrode. J. of Electroanalytical Chem. 570: 83 - 90. Wang, J. (1985): Stripping analysis: Principles, Instrumentation and Application. USA: VCH Publishers. Wang, J., Cai, X., Fernandes, J. R., Ozsoz, M. and Grant, D. H., (1997). Adsorptive Potentiometric Stripping Analysis of Trace Tomoxifen at a Glassy Carbon Electrode, Talanta. 45: 273 - 278. Wang, J. (1988). Electroanalytical Technique in Clinical Chemistry and Laboratory

Medicine. USA: VCH publisher. Wang, J. (2000). Analytical Electrochemistry. USA: WILEY-VCH. Weast, R.C and Astle, M.J. (1981). Handbook of Chemistry and Physics. 61 th Ed. Flourida: CRC Press. Wasiak, W, Cissewska, W. and Ciszewski, A. (1996). Hair Analysis Part I: Differentrial Pulse Anodic Stripping Voltammetry Determination of Lead, Cadmium, Zinc and Copper in Human Hair Samples of Persons in Permanent Contact With a Polluted Workplace Environment. Anal. Chim. Acta. 335: 201 207. Weast, R.C and Astle, M.J. (1987). CRC Handbook of Data On Organic

Compounds, Vol II. Flourida: CRC Press. Wei, R.D., Chang, S.Ch. and Lee, S.S. (1980). High Pressure Liquid Chromatography Determination of Aflatoxins in Soy Sauces anf Fermented Soybean Paste. J. Assoc. Off. Anal. Chem. 63:1269- 1274.

252 White, C.E. and Argauer, R.J. (1970). Fluorescence Analysis- A Practical Approach. New York: Marcell Dekker. Wilson, T.J. and Romer, T.R (1991). Use of the Mycosep Multifunctional Clean-up Column for Liquid Chromatography Determination of Aflatoxins in Agriculture Products. J. Assoc. Off. Anal. Chem.74 : 951 - 956. Wogan, G.N., Hecht, S. S., Felton, J. S., Conney, A.H. and Loeb, L.A. (2004). Environmental and Chemical Carcinogenesis. Seminars In Cancer Biology. 14: 473 - 486. Woolever, C.A. and Dewald,H.D. (2001). Differential Pulse Anodic Stripping Voltammetry of Barium and Lead in Gunshot Residues. Forensic Sci Int. 117: 185-190. World Health Organisation (1987) in: IARC Monograph on the Evaluation of Carcinogenic Risk to Human, WHO, Lyon, pp 82 Supl. I. Wring S. A., Hart J.P. and Birck B.J. (1991). Voltammetric Behaviour of Screenprinted Carbon Electrode, Chemically Modified With Selected Mediatiors and Their Applications as Sensor for Detection of Reduced Gluthathione. Analyst.

116 : 123-129. www.ansci.cornell.edu/toxicagents/aflatoxin. Quoted date - 30 th. July 2002. www.ccohs.ca/oshanswers/chemicals. Quoted date – 5 th February 2006 www.copdockmill.co.uk/aflatoxin Quoted date – 26 th August 2001 Ximenes, M.I.N., Rath, S. and Reyes, F.G.R. (2000). Polarographic Determination of Nitrate in Vegetable. Talanta. 51: 49 - 56.

253 Xiulan S., Xiaolian, Z., Jian, T., Xiaohong, G., jun, Z. and Chu, F.S. (2006). Development of an Immunochromatographic Assay for Detection of Aflatoxin B1 in Foods. Food Control. 17: 256 - 262. Yardimer, C. and Ozaltin, N. (2001). Electrochemical Studies and Differential Pulse Polarographic Analysis of Lansoprazole in Pharmaceuticals. Analyst. 126: 361366. Yardimer, C. and Ozaltin, N. (2004). Electrochemical Studies and Square-wave Voltammetric Determination of Fenofibrate in Pharmacetical Formulations.

Anal. Bioanal. Chem. 378: 495 - 498. Yaacob, M.H. (1993). Polarographic Determination of Sn(II) in

Radiopharmaceutical Samples. University of Manchester, Institute of Science and Technology (UMIST), United Kingdom. MSc Thesis. Yilmaz, S., Uslu, B. and Ozkan, S.A. (2001). Anodic Oxidation of Etodolac and its Square Wave and Differential Pulse Voltammetric Determination in Pharmaceuticals and Human Serum. Talanta. 54: 351 - 360. Youmens, H.L. (1973). Statistics for Chemistry. USA: Bells and Howell Publishers. Yousef, A.E and Marth, E.H. (1985). Rapid Reverse Phase Liquid Chromatography Determination of AFM1 in Milk. J. Assoc. Off. Anal. Chem. 68: 462 - 465. Zahir, M.H. and Abd. Ghani, S. (1997). Fabrication of Directly Polymerized 4vinylpyridine onto a Pencil 2B Graphite Paste Electrode for Glucose Monitoring, Anal Chim Acta, 354: 351 - 358.

254 Zayed, S.I.M. and Habib, I.H.I. (2005). Adsorptive Stripping Voltammetric Determination of Triprolidine Hydrochloride in Pharmaceutical Tablets. I1 Farmaco. 60(6-7): 621 - 625. Zhang, Z.Q., Zhang, H. and H,e G. F. (2002). Preconcentration with Membrane Cell and Adsorptive Polarographic Determination of Formaldehyde in Air. Talanta.

57: 317 - 322. Zhang, Z.Q, Li, Y.F., He, X.M. and Zhang, H. (1996). Electroanalytical Characteristics of Enoxican and Their Analytical Application. Talanta.43: 635 641. Zhao, C., Pan, Y., Su, Y., Zhang, Z., Guo, Z. and Sun, L. (2003). Determination of EDTA Species in Water by Square-wave Voltammetry using a Chitosan-coated Glassy Carbon Electrode. Water Research. 37 : 4270 - 4274. Zima, J., Barek, J., Moriera, J.C., Mejstrik, V. and Fogg, A.G. (2001). Electrochemical Determination of Trace Amounts of Environmentally Important Dyes. Fresenius. J. Anal. Chem. 369: 567 - 570. Zimmermann, S., Menzel A., Bernaz Z., Eckhardt J-D., Stubens D., Alt F., Messerschmidt H., Taraschewski H. and Sures B.(2001). Trace Analysis of Platinum in Biological Samples: A Comparison Between Sector field ICP-MS and Adsorptive Cathodic Stripping Voltammetry Following Different Digestion Procedures. Anal. Chim. Acta. 439 : 203 - 209.

255

APPENDIX A

Relative fluorescence of aflatoxins in different solvents

Aflatoxin

Methanol

Ethanol

Chloroform

B1

0.6 (430 nm)

1.0 (430 nm)

0.20 (413 nm)

B2

5.3 (430 nm)

2.7 (430 nm)

0.25 (413 nm)

G1

1.0 (450 nm)

1.4 (450 nm)

6.2 (430 nm)

G2

8.7 (450 nm)

4.7 (450 nm)

6.8 (430 nm)

256

APPENDIX B

257

APPENDIX C

258

APPENDIX D

Calculation of concentration of aflatoxin stock solution Formula used for calculation of concentration of aflatoxin stock solution by UVVIS spectrometric technique [Aflatoxin] / µg/ml

=

Abs x

MW ε

x

1000

Where Abs

=

absorbance value

MW

=

molecular weight

ε

=

molar absorbtivity (cm-1 M-1)

Parameters used in this calculation;

Aflatoxin

MW

AFB1

312

AFB2

314

AFG1 AFG2

Solvent

ε

Λ (nm)

19,800

353

20,900

355

328

17,100

355

330

18,200

357

Benzene:acetonitrile (98:2)

Example: AFB1 Abs = 0.640 [AFB1] =

MW = 312 0.640 x 312 x 1000 19,800

ε = 19,800 =

10.08 µg / ml

= 10.08 ppm

259

APPENDIX E Extraction procedure for aflatoxins in real samples Extraction procedure for aflatoxins in real samples: Chemistry Dept. Penang Branch, Ministry of Science, Technology and Innovation; 1. Groundnut with shell: remove shell of entire sample. Coarse grind. Remove 50 g and regrind this portion to finer size for drawing analytical sample. 2. Transfer 25 gm analytical sample into a waring blender and add 125 ml of MeOH-0.1N HCl (100:25) and blend for 2 min (timing). Stand for 5 min (timing). 3. Filter through whatman No. 1 paper or equivalent. Complete filtration as soon as possible 4. Collect 50 ml of filtrate in a stoppered container. (Equivalent weight: 125 ml / 25 g sample) 5. Pipette 20 ml of filtrate into a stoppered or screw cap bottle (Equivalent weight: 20 ml / 4 g sample) 6. Add exactly 20 ml of 15% ZnSO4 solution. Stopper or cap tightly and shake vigorously for 30 sec. Filter through diatomaceous earth into another container. (Equivalent weight: 40 ml / 4 g sample)

7. Pipette 20 ml of this filtrate into a small separating funnel and add exactly 5 ml of chloroform. Stopper or cap tightly and shake vigorously for 30 sec. (Equivalent weight: 20 ml / 2 g sample).Equivalent weight: 5 ml chloroform / 2 g sample) 8. Stand for a few minute to let layers separate. After separated, draw chloroform layer into a suitable container and pipette 1 ml extract accurately into amber bottle for preparation of final sample before injecting into voltammetric cell.

260

APPENDIX F

Calculation of individual aflatoxin in groundnut sample

Aflatoxin, ng/g (ppb) =

P x C x 1000 µl x 125 ml x 1 P’ 100 µl V W where; P = peak height of sample (nA) P’= peak height of standard (after substract with peak height of sample) (nA) C = amount of aflatoxin injected into voltammetric cell (ng) V = effective volume; = 20 x volume of chloroform used for sample preparation = 20 x 1 = 2 ml 2 total volume of chloroform added 2 5 W = weight of sample (25 g)

===> P x C x 1000 µl x 125 ml x 1 100 µl 2 ml 25 g P’ ===> P x C x 25 ( for injected volume = 100 µl) P’

261 For other injection volume; Volume (µl)

Formula

200

P x C x 12.5 P’

300

P x C x 8.33 P’

400

P x C x 6.25 P’

Notes; From first equation; 1000 µl

=

Volume of final solution prepared in BRB at pH 9.0

100 µl

=

Injection volume of sample

125 ml

=

Volume of solvent for mixing and blending groundnut

262

APPENDIX G

Cyclic voltammograms of AFB1, AFG1 and AFG2 with increasing of their concentrations

Figure G-1 Effect of increasing AFB1 concentrations on the Ip of cathodic cyclic voltammetric curves in BRB at pH 9.0. (a) 1.30 µM, (b) 2.0 µM (c) 2.70 µM and (d) 3.40 µM. Parameters conditions are Ei = 0, Elow = -1.5 V, Ehigh = 0, scan rate = 200 mV/s.

263

Figure G-2 Effect of increasing AFG1 concentrations on the Ip of cathodic cyclic voltammetric curves in BRB at pH 9.0. (a) 1.30 µM, (b) 2.0 µM (c) 2.70 µM and (d) 3.40 µM. Parameters conditions are Ei = 0, Elow = -1.5 V, Ehigh = 0, scan rate = 200 mV/s.

Figure G-3 Effect of increasing AFG2 concentrations on the Ip of cathodic cyclic voltammetric curves in BRB at pH 9.0. (a) 1.30 µM, (b) 2.0 µM (c) 2.70 µM and (d) 3.40 µM. Parameters conditions are Ei = 0, Elow = -1.5 V, Ehigh = 0, scan rate = 200 mV/s.

264

APPENDIX H

Dependence of the peak heights of AFB1, AFG1 and AFG2 on their concentrations

120

Ip (nA)

90 60 30

y = 22.451x + 10.989 R2 = 0.9899

0 1

1.5

2

2.5

3

3.5

4

[AFB1] / uM

Figure H-1 Dependence of the Ip of AFB1 on concentration of AFB1 in BRB solution at pH 9.0.

265

120

Ip (nA)

90 60 y = 24.756x + 13.072

30

R2 = 0.9812

0 1

1.5

2

2.5

3

3.5

4

[AFG1] / uM

Figure H-2 Dependence of the Ip of AFG1 on concentration of AFG1 in BRB solution at pH 9.0.

120 100

Ip (nA)

80 60 40 y = 28.757x + 1.4407 R2 = 0.9914

20 0 1

1.5

2

2.5

3

3.5

4

[AFG2] / uM

Figure H-3 Dependence of the Ip of AFG2 on concentration of AFG2 in BRB solution at pH 9.0.

266

APPENDIX I

Repetitive cyclic voltammograms and their peak height of AFB2, AFG1 and AFG2 in BRB at pH 9.0

Figure I-1 Repetitive cathodic cyclic voltammograms of 1.3 µM AFB1 in BRB solution at pH 9.0. Parameter conditions are Ei = 0, Elow = -1.5 V, Ehigh = 0 and scan rate = 200 mV/s

267

70 60 50 Ip (nA)

40 30 20 10 0

1

2

3

4

5

No of cycle

Figure I-2 Increasing Ip of AFB1 cathodic peak obtained from repetitive cyclic voltammetry

Figure I-3 Repetitive cathodic cyclic voltammograms of 1.3 µM AFG1 in BRB solution at pH 9.0. Parameter conditions are Ei = 0, Elow = -1.5 V, Ehigh = 0 and scan rate = 200 mV/s

268

80 70

Ip (nA)

60 50 40 30 20 10 0 1

2

3

4

5

No of cycle

Increasing Ip of AFG1 cathodic peak obtained from repetitive cyclic Figure I-4 voltammetry

Figure I-5 Repetitive cathodic cyclic voltammograms of 1.3 µM AFG2 in BRB solution at pH 9.0. Parameter conditions are Ei = 0, Elow = -1.5 V, Ehigh = 0 and scan rate = 200 mV/s

269

70 60 50 Ip (nA)

40 30 20 10 0

1

2

3

4

5

No of cycle

Figure I-6 Increasing Ip of AFG2 cathodic peak obtained from repetitive cyclic voltammetry

270

APPENDIX J

Voltammetric plot E p-log υ for the reduction of AFB1, AFG1 and AFG2 in BRB at pH 9.0

1350

Ep (-mV)

1340 1330 1320 1310 1300 1290 1280 1270

y = 61.415x + 1171.6 R2 = 0.9987

1260 1250 1240 1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

log v

Figure J-1 The voltammetric plot Ep – log υ for the reduction of 1.3 µM AFB1 in BRB solution at pH 9.0

271

Ep (-m V)

1280 1270 1260 1250 1240 1230 1220 1210

y = 48.484x + 1134.8 R2 = 0.9978

1200 1190 1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

log v

Figure J-2 The voltammetric plot Ep – log υ for the reduction of 1.3 µM AFG1 in BRB solution at pH 9.0

1290 1280 1270 Ep (-mV)

1260 1250 1240 1230 y = 49.297x + 1146 R2 = 0.9984

1220 1210 1200 1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

log v

The voltammetric plot Ep – log υ for the reduction of 1.3 µM AFG2 in Figure J-3 BRB solution at pH 9.0

272

APPENDIX K

Plot of peak height versus scan rate for AFB1, AFG1 and AFG2 in BRB at pH 9.0

80 70

Ip (nA)

60 50 40 30 20 10 0 0

100

200

300

400

500

600

v ( mV/sec)

Figure K-1

Plot of Ip versus scan rate (υ) for 1.3 µM AFB1 in BRB at pH 9.0

Ip (n A)

273

110 100 90 80 70 60 50 40 30 20 10 0 0

100

200

300

400

500

600

v ( mV/s )

Figure K-2

Plot of Ip versus scan rate (υ) for 1.3 µM AFG1 in BRB at pH 9.0

70 60

Ip (nA)

50 40 30 20 10 0 0

100

200

300

400

500

600

v ( mv/sec)

Figure K-3

Plot of Ip versus scan rate (υ) for 1.3 µM AFG2 in BRB at pH 9.0

274

APPENDIX L

Voltammograms of AFB2 with increasing concentrations

Figure L-1

Cathodic stripping voltammograms of increasing concentration of AFB2

in (a) BRB at pH 9.0, (b) 0.02 µM (c) 0.06 µM (d) 0.10 µM (e) 0.14 µM, (f) 0.18 µM (g) .22 µM (h) 0.26 µM and (i) 0.32 µM. Parameter conditions; Ei = -1.0 V, Ef = -1.4 V, Eacc = -0.60 V, t acc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV.

275

APPENDIX M

Voltammograms of 0.1 µM and 0.2 µM AFB2 obtained on the same day measurements

Figure M-1 Cathodic stripping voltammograms of 0.1µM AFB2 obtained in the same day (n= 8) with RSD = 2.83% Eacc = -6.0 V, tacc= 80 s, υ = 50 mV/s and pulse amplitude = 80 mV

Figure M-2 Cathodic stripping voltammograms of 0.2 µM AFB2 obtained in the same day (n= 8) with RSD = 0.72%. Eacc = -6.0 V, t acc= 80 s, υ = 50 mV/s and pulse amplitude = 80 mV

276

APPENDIX N

Voltammogramms of AFB2 at inter-day measurements

Figure N-1 Cathodic stripping voltammograms of 0.1µM AFB2 obtained at day 1 ( n= 8) with RSD = 2.83% .Eacc = -6.0 V, tacc= 80 s, υ = 50 mV/s and pulse amplitude = 80 mV

277

Figure N-2 Cathodic stripping voltammograms of 0.1µM AFB2 obtained at day 2 (n= 8) with RSD = 2.39% Eacc = -6.0 V, tacc= 80 s, υ = 50 mV/s and pulse amplitude = 80 mV

Figure N-3 Cathodic stripping voltammograms of 0.1µM AFB2 obtained at day 3 (n= 8) with RSD = 1.31% Eacc = -6.0 V, tacc= 80 s, υ = 50 mV/s and pulse amplitude = 80 mV

278

APPENDIX O

F test for robustness and ruggedness tests a) Two tailed F test was used to observe any significant different of variance by using small variation of a few important parameters such pH of buffer solution, Eacc and tacc.. Optimum parameters

Variation

pH of BRB = 9.0

8.5 and 9.5

Eacc = -0.60 V

-0.59 V and -0.61 V

tacc = 80 s

75 and 85 s

0.1 µM AFB2 (n =5): For optimum and small variation in pH of BRB (as an example) pH

n

Ip

SD

9.0

5

60.62

0.88

8.5

5

58.82

0.79

F = (0.88)2 / (0.79)2 = 0.7744/ 0.6241 = 1.24 (< F tabulated at 95 % confidential level; 9.60). No significant different for pH 9.0 and 8.5 at 95% confidential level.

279

pH

n

Ip

SD

9.0

5

60.62

0.88

9.5

5

59.30

0.44

F = (0.88)2 / (0.44)2 = 0.7744 / 0.1936 = 4.00 (< F tabulated at 95 % confidential level; 9.60). No significant different for pH 9.0 and 9.5 at 95% confidential level. b)

Two tailed F test was used to observe any significant different of variance by

using different voltammetric analyser which are BAS and Metrohm under optimum parameters (AFB1 as an example). AFB1 Voltammetric analyser

n

Ip

SD

Metrohm

5

59.88

0.94

BAS

5

58.28

2.79

F(4,4) at 95% confidence level = 9.60 F = (2.79)2 / (0.94)2 = 7.78 / 0.88 = 8.84 (< F tabulated; 9.60) No significant difference between the results obtained for 0.1 µM AFB1 by two types of voltammetric analyser.

280

APPENDIX P

Voltammograms of AFB1 with increasing concentration

Figure P-1

Cathodic stripping voltammograms of increasing concentration of AFB1

in (a) BRB at pH 9.0, (b) 0.02 µM (c) 0.04 µM (d) 0.14 µM (e) 0.18 µM, (f) 0.22 µM (g) 0.26 µM (h) 0.30 µM and (i) 0.32 µM. Parameter conditions; Ei = -1.0 V, Ef = -1.4 V, Eacc = -0.60 V, t acc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV.

281

APPENDIX Q

Voltammograms of AFG1 with increasing concentrations

Figure Q-1

Cathodic stripping voltammograms of increasing concentration of AFG1

in (a) BRB at pH 9.0, (b) 0.02 µM, (c) 0.04 µM, (d) 0.06 µM, (e) 0.08 µM, (f) 0.10 µM, (g) 0.14 µM, (h) 0.18 µM, (i) 0.26 µM (j) 0.30 µM and (k) 0.32 µM. Parameter conditions; Ei = -0.95 V, Ef = -1.4 V, Eacc = -0.60 V, t acc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV.

282

APPENDIX R

Voltammograms of AFG2 with increasing concentrations

Figure R-1

Cathodic stripping voltammograms of increasing concentration of AFG2

in (a) BRB at pH 9.0, (b) 0.02 µM, (c) 0.04 µM, (d) 0.06 µM, (e) 0.10 µM, (f) 0.14 µM, (g) 0.18 µM, (h) 0.26 µM, (i) 0.28 µM and (j) 0.30 µM. Parameter conditions; Ei = -1.0 V, Ef = -1.4 V, Eacc = -0.60 V, t acc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV.

283

APPENDIX S

LOD determination according to Barek et al. (2001a) By standard addition of lower concentration of analyte (AFB1, AFB2, AFG1 and AFG2) until obtaining the sample response that is significantly difference from blank sample

a) AFB1;

0.5 x 10-8 M or 1.56 ppb gave Ip = 4.3 nA at Ep = -1.250 V

Figure S-1

Voltammogram of 0.5 x 10 -8 M of AFB1 in BRB at pH 9.0

284 b) AFB2;

0.78 x 10-8 M or 2.5 ppb gave Ip = 5.7 nA at Ep = -1.260 V

Figure S-2

Voltammogram of 0.78 x 10-8 M of AFB2 in BRB at pH 9.0

c) AFG1;

1.0 x 10-8 M or 3.28 ppb gave Ip = 4.34 nA at Ep = -1.160 V

Figure S-3

Voltammogram of 1.0 x 10 -8 M of AFG1 in BRB at pH 9.0

285

d) AFG2;

0.76 x 10-8 M or 2.5 ppb gave Ip = 6.19 nA at Ep = -1.190 V

Figure S-4

Voltammogram of 0.76 x 10-8 M of AFG2 in BRB at pH 9.0

From the results, LOD for AFB1, AFB2, AFG1 and AFG2 are 0.5 x 10-8 M (1.56 ppb), 0.78 x 10-8 M (2.50 ppb), 1.0 x 10-8 M (3.28 ppb) and 0.76 x 10-8 M (2.50 ppb), respectively.

286

APPENDIX T

LOD determination according to Barek et al. (1999) The limit of detection is calculated as threefold standard deviation from seven analyte determinations at the concentration corresponding to the lowest point on the appropriate calibation curve. AFB1 (as an example); concentration for lowest point on calibration curve is 2.0 x 10-8 M. Ip values from seven determinations of this concentration; 11.76, 12.00, 12.40, 11.80, 11.85, 11.92, 11.92 Mean = 11.95

Standard deviation = 0.2142

0.2142 x 3 = 0.642 x 10-8 M or 2.00 ppb LOD for determination of AFB1 = 0.64 x 10-8 M or 2.00 ppb.

287

APPENDIX U

LOD determination according to Zhang et al. (1996) The LOD is the concentration giving a signal equal to three times the standard deviation of the blank signal divided by slope from calibration curve. AFB1 as an example Regression equation for calibration curve is y = 5.4363x + 3.7245 Slope = 5.4363 Ip for blank from seven measurements; 40.86, 42.24, 40.22, 38.53, 42.52, 41.04, 41.64 standard deviation of blank (SDblk) = 1.3533 3SDblk / m = (3 x 1.3533) / 5.4363 = 0.7468 x 10-8 M or 2.35 ppb

LOD for determination of AFB1 = 0.75 x 10-8 M or 2.35 ppb.

288

APPENDIX V

LOD determination according to Miller and Miller (1993) LOD is the concentration giving a signal three times the standard error plus the yintercept divided by the slope of calibration curve Regression equation for peak height of analyte with their concentrations is; yi = mx + b where;

yi = peak height m = slope b = y-intercept

y value for calculation of standard error is y’i = mx + b where x is the concentration of analyte. Standard error is calculated based on following equation; Sy/x = √( ∑ (yi – yi’)2 ) / n -2 LOD = (3 x Sy/x ) / m

289

As example, LOD for AFB1 is calculated as below; Regression equation for AFB1; y = 5.4363x + 3.7245

[AFB1] = x

Peak height = y

y i’ = mx + b

y – y i’

2

11.86

14.597

-2.737

7.491

4

23.77

25.470

-1.70

2.89

6

36.34

36.342

-0.002

4 x 10-6

10

60.4

58.088

2.312

5.345

14

82.12

79.833

2.287

5.230

18

103

101.578

1.422

2.022

22

125.1

123.323

1.777

3.158

26

145.6

145.068

0.532

0.283

30

165.2

166.814

-1.614

2.605

32

175.4

177.686

-2.286

5.226

∑ (yi – yi’ )2

34.250

(x in 10 -8 M and peak height is in nA) n = 10, m = 5.4363, ∑ (yi – yi’)2 = 34.250 Sy/x = √34.250/ 8 = 2.069 LOD = (3 x 2.069) / 5.4363 = 1.142 x 10-8 M or 3.56 ppb ====> LOD for AFB1 is 1.14 x 10-8 M or 3.56 ppb.

(y – yi’)2

290

APPENDIX W

ANOVA test (Youmens, 1973) Table W-1

LOD (in ppb) of aflatoxins obtained from three different method

Aflatoxin

B1

B2

G1

G2

Totals

1

1.56

2.50

3.28

2.50

9.84

2

2.00

2.80

3.50

3.02

11.32

3

2.35

2.86

3.60

2.84

11.65

Totals

5.91

8.16

10.38

Method

8.36

32.81

Four sums of squares were calculated in order to make up the ANOVA table. They are total, sample, method and error sums of squares. The sums of squares were calculated in five steps as follows; a) Calculation of C = (∑ Xij)2 / kn = (32.81)2 / 12 = 89.71 b) Calculation of total sum of squares = ∑ (Xij)2 – C = 93.63 – 89.71 = 3.92 c) Calculation of sample or block sum of squares = 1 ∑2j – C = 1 x 279.15 – 89.71 n 3 = 3.34 d) Calculation of method or process sum of squares =1 ∑2i– C k = 1 x 360.69 – 89.71 = 0.46 4

291 e) Calculation of error sum of squares Error sum of squares = total sum of squares – (block sum of squares + process sum of squares) = 3.92 – (3.34 + 0.46) = 0.12 The ANOVA table was constructed as follows:

Table W-2

Analysis of variance

Source

Degrees of freedom (f)

Sum of squares (SS)

Mean squares (SS / f = MS)

Total

kn – 1 = 11

3.92

Block

n–1=2

3.34

Process

k–1=3

0.46

0.153

Error

kn – n – k + 1 = 6

0.12

0.02

f) Calculation of F F = process mean square / error mean square = 0.153 / 0.02 = 7.65 g) Test of null hypothesis Tabular F from Fisher’s F table is 8.94 for f1 = 6 and f2 = 3. This is larger than the calculated value of F. The hypothesis was not disproved, hence the experiment indicates that the methods are not giving significantly different of LOD of aflatoxins at 95% probability level. This ANOVA test indicates that all three methods can be selected in determination of LOD of aflatoxins using proposed technique.

292

APPENDIX X

Peak height of AFB2 obtained from modification of mercury electrode with PLL

Ip of 10 ppb all aflatoxins in BRB at pH 9.0 in presence and absence of Table X-1 poly-L-lysine (PLL)

No PLL

With PLL

Aflatoxin Ip (nA)

E p (V)

Ip (nA)

Ep (V)

AFB1

15.70

-1.26

10.22

-1.22

AFB2

14.83

-1.26

12.20

-1.26

AFG1

18.77

-1.19

16.20

-1.19

AFG2

17.60

-1.22

13.90

-1.22

60

Ip (nA)

50 40

No PLL

30

W ith PLL

20 10 0 5

6

9

11

pH of BRB

Figure X-1 Ip of 0.1 µM AFB2 (31.4 ppb) in different pH of BRB with and without PLL coated on mercury electrode.

293

APPENDIX Y

SWSV voltammograms of AFB1, AFB2, AFG1 and AFG2 in BRB at pH 9.0

Figure Y-1 SWS voltammograms of AFB1 in BRB at pH 9.0 (n =10). Experimental parameters; Ei = -1.0 V, Ef = -1.4 V, Eacc = -0.8 V, tacc = 100 s, υ = 3750 mV/s, frequency = 125 Hz, voltage step = 0.03 s and amplitude = 50 mV. Blank is represented by broken line.

Figure Y-2 SWS voltammograms of AFB2 in BRB at pH 9.0 (n=10). All experimental conditions are the same as in the Figure Z-2.

294

Figure Y-3 SWS voltammograms of AFG1 in BRB at pH 9.0 (n=10). All experimental conditions are the same as in the Figure Z-2 except for Ei = -0.95 V.

Figure Y-4 SWSV voltammograms of AFB2 in BRB at pH 9.0 (n=10). All experimental conditions are the same as in the Figure Z-2.

295

APPENDIX Z

SWSV voltammograms of 0.1 µM of AFB1, AFB2, AFG1 and AFG2 in BRB at pH 9.0

Figure Z-1 SWSV voltammograms of 0.10 µM of AFB1 (n =3) in BRB at pH 9.0. Ei = -1.0 V, Ef = -1.4 V, Eacc = -0.8 V, t acc = 100 s, υ = 3750 mV/s, frequency = 125 Hz, voltage step = 0.03 V and amplitude = 50 mV.

Figure Z-2 SWSV voltammograms of 0.10 µM of AFB2 (n = 3) in BRB at pH 9.0. All experimental parameters are the same as in Figure Z-1.

296

Figure Z-3 SWSV voltammograms of 0.10 µM of AFG1 (n=3) in BRB at pH 9.0. All experimental parameters are the same as in Figure Z-1 except for Ei = -0.95 V.

Figure Z-4 SWSV voltammograms of 0.10 µM of AFG2 (n =3) in BRB at pH 9.0. All experimental parameters are the same as in Figure Z-1.

297

APPENDIX AA UV-VIS spectrums of 10 ppm AFB1, AFB2, AFG1 and AFG2 stock solutions

Figure AA-1 UV-VIS spectrums (n=3) of 10 ppm AFB1 in benzene: acetonitrile (98%)

Figure AA-2 UV-VIS spectrums (n=3) of 10 ppm AFB2 in benzene: acetonitrile (98%)

298

Figure AA-3 UV-VIS spectrums (n=3) of 10 ppm AFG1 in benzene: acetonitrile (98%)

Figure AA-4 UV-VIS spectrums (n=3) of 10 ppm AFG2 in benzene: acetonitrile (98%)

299

APPENDIX AB

Voltammograms of AFB1, AFB2, AFG1 and AFG2 obtained from 0 to 6 months storage time in the cool and dark conditions

Figure AB-1 Voltammograms of 0.10 µM AFB1 in BRB at pH 9.0 obtained from difference storage time of 0 to 6 months in the dark and cool conditions. DPCSV parameter conditions: Ei = -1.0 V, E f = -1.4 V, Eacc = -0.6 V, tacc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV.

300

Figure AB-2 Voltammograms of 0.10 µM AFB2 in BRB at pH 9.0 obtained from difference storage time of 0 to 6 months in the dark and cool conditions. DPCSV parameter conditions are the same as in Figure AB-1.

Figure AB-3 Voltammograms of 0.10 µM AFG1 in BRB at pH 9.0 obtained from difference storage time of 0 to 6 months in the dark and cool conditions. DPCSV parameter conditions are the same as in Figure AB-1 except for Ei = -0.95 V.

301

Figure AB-4 Voltammograms of 0.10 µM AFG2 in BRB at pH 9.0 obtained from difference storage time of 0 to 6 months in the dark and cool conditions. DPCSV parameter conditions are the same as in Figure AB-1.

302

APPENDIX AC

Voltammograms of AFB1, AFB2, AFG1 and AFG2 obtained from 0 to 8 hours exposure time

Figure AC-1 Voltammograms of 0.10 µM AFB1 in BRB at pH 9.0 exposed to normal laboratory conditions from 0 to 8 hrs. Experimental conditions: Ei = -1.0 V, Ef = -1.4 V, Eacc = -0.60 V, t acc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV.

303

Figure AC-2 Voltammograms of 0.10 µM AFB2 in BRB at pH 9.0 exposed to normal laboratory conditions from 0 to 8 hrs. Experimental conditions are the same as in Figure AC-1.

Figure AC-3 Voltammograms of 0.10 µM AFG1 in BRB at pH 9.0 exposed to normal laboratory conditions from 0 to 8 hrs. Experimental conditions are the same as in Figure AC-1 except for Ei = -0.95 V.

304

Figure AC-4 Voltammograms of 0.10 µM AFG2 in BRB at pH 9.0 exposed to normal laboratory conditions from 0 to 8 hrs. Experimental conditions are the same as in Figure AC-1.

305

APPENDIX AD

UV-VIS spectrums of AFB2 and AFG2 in BRB at pH 6.0 and 11.0

Figure AD-1 UV-VIS spectrums of 1.0 ppm AFB2 in BRB at pH 6.0 from 0 to 3 hrs exposure time

Figure AD-2 UV-VIS spectrums of 1.0 ppm AFB2 in BRB at pH 11.0 from 0 to 3 hrs exposure time

306

Figure AD-3 UV-VIS spectrums of 1.0 ppm AFG2 in BRB at pH 6.0 from 0 to 3 hrs exposure time

Figure AD-4 UV-VIS spectrums of 1.0 ppm AFG2 in BRB at pH 11.0 from 0 to 3 hrs exposure time

307

APPENDIX AE

Voltammograms of AFB1 and AFG1 in 1.0 M HCl and 1.0 M NaOH

Figure AE-1 DPCSV voltammograms of AFB1 in 1.0 M HCl from 0 to 6 hours

Figure AE-2 DPCSV voltammograms of AFB1 in 1.0 M NaOH from 0 to 6 hours.

308

Figure AE-3 DPCSV voltammograms of AFG1 in 1.0 M HCl from 0 to 6 hours.

Figure AE-4 DPCSV voltammograms of AFG1 in 1.0 M NaOH from 0 to 4 hours.

309

APPENDIX AF

DPCS voltammograms of real samples added with various concentrations of AFG1

(i)

(ii)

(iii)

Figure AF-1 DPCSV voltammograms of real samples (b) added with 3 ppb (i), 9 ppb (ii) and 15 ppb (iii) AFG1 obtained in BRB at pH 9.0 (a) as the blank on the first day measurement.

310

APPENDIX AG

SWSV voltammograms of real samples added with various concentrations of AFB1

(i)

(ii)

(iii)

Figure AG-1 SWSV voltammograms of real samples (b) added with 3 ppb (i), 9 ppb (ii) and 15 ppb (iii) AFB1 obtained in BRB at pH 9.0 (a) as the blank on the first day measurement.

311

APPENDIX AH

Percentage of recoveries of 3 ppb and 9 ppb of all aflatoxins in real samples obtained by SWSV method.

120

% recovery

100 80

AFB1 AFB2

60

AFG1 AFG2

40 20 0 Da y 1

Day 2

Da y 3

Figure AH-1 Percentage of recoveries of 3 ppb of all aflatoxins in real samples obtained by SWSV method for one to three days of measurements.

120

% recovery

100 80

AFB1 AFB2

60

AFG1 AFG2

40 20 0 Da y 1

Day 2

Da y 3

Figure AH-2 Percentage of recoveries of 9 ppb of all aflatoxins in real samples obtained by SWSV method for one to three days of measurements.

312

APPENDIX AI

Calculation of percentage of recovery for 3.0 ppb AFG1 added into real sample.

From voltammograms of real sample added with 3.0 ppb AFG1;

Peak height = 5.85 nA, 6.53 nA, 6.09 nA

Average = 6.16 nA

Peak height for 10 ppb AFG1 in BRB pH 9.0 = 20.60 nA*. So for 1 ppb = 2.060 nA For peak height = 6.16 nA ===> (6.16 / 2.060) x 1.0 ppb = 2.99 ppb Injected AFG1 = 3.0 ppb From the above calculation, found AFG1 = 2.99 ppb Recovery (%) = (2.99 / 3.0) x 100 % = 99.67%. Summary: AFG1 added = 3. 0 ppb AFG1 found = 2.99 ppb % recovery of AFG1 in real sample = 99.67 % *

Notes; For other aflatoxins, the peak heights for 10 ppb of AFB1, AFB2 and AFG2 in

BRB pH 9.0 is 21.93 nA, 21.33 nA and 20.23 nA respectively.

313

APPENDIX AJ

HPLC chromatograms of real samples: S10 and S07

Figure AJ-1 HPLC chromatogram for S10 which contains 36.00 ppb total aflatoxins.

Figure AJ-2 HPLC chromatogram for S07 which contains 3.67 ppb total aflatoxins.

314

APPENDIX AK

Calculation of aflatoxin in real sample; S13 1 st analysis From voltammograms of real sample ; Peak height = 1.78 nA, 1.80 nA, 1.79 nA

Average = 1.79 nA

From voltammograms of real sample added with 10 ppb AFB1 standard solution. Peak height = 22.5 nA, 22.6 nA, 22.8 nA

Average = 22.63 nA

From equation as stated in Appendix F; Aflatoxin content = [(1.79) / (22.63 – 1.79)] x 12.5 = 10 .74 ppb

2 nd analysis From voltammograms of real sample ; Peak height = 1.80 nA, 1.79 nA, 1.82 nA

Average = 1.80 nA

From voltammograms of real sample added with 10 ppb AFB1 standard solution. Peak height = 22.7 nA, 22.5 nA, 22.8 nA

Average = 22.67 nA

From equation as stated in Appendix F; Aflatoxin content = [(1.80) / (22.67 – 1.80)] x 12.5 = 10 .78 ppb Average for duplicate analysis = (10.74 + 10.78) / 2 = 10.74 ppb Total aflatoxins content in S13 = 10.76 ppb

315

APPENDIX AL List of papers presented or published to date resulting from this study 1.

Yaacob, M.H., Mohd. Yusoff, A.R. and Ahamad, R. Cylic voltammetry study of

AFB2 at the mercury electrode. Paper presented at SKAM -16, Kucing, Sarawak, 9 – 11th September 2003. 2.

Yaacob, M.H., Mohd. Yusoff, A.R. and Ahamad, R. Cylic voltammetry study of

AFB2 at the mercury electrode. Malaysian J. Anal. Sci. In press. 3.

Yaacob, M.H., Mohd. Yusoff, A.R. and Ahamad, R. Differential pulse stripping

voltammetric technique for determination of AFB2 at the mercury electrode. J. Collect.

Czech. Chem. Common. In press 4.

Yaacob, M.H., Mohd. Yusoff, A.R. and Ahamad, R. Stability studies of

aflatoxin G1 (AFG1) using differential pulse stripping voltammetric technique. Paper presented at Symposium Life Science II, USM Penang. 31st to 3rd April 2004. 5.

Yaacob, M.H., Mohd. Yusoff, A.R. and Ahamad, R. Developement of

differential pulse stripping voltammetric (DPCSV) technique for determination of AFG1 at the mercury electrode. Chemical Analysis (Warsaw). In press 6.

Yaacob, M.H., Mohd. Yusoff, A.R. and Ahamad, R. Cyclic voltammetry study

of AFG1 at the mercury electrode. Paper presented at KUSTEM 3rd Annual Seminar on Sustainability Science and Management, Kuala Terengganu, Terengganu. 4 – 5th May 2004. 7.

Yaacob, M.H., Mohd. Yusoff, A.R. and Ahamad, R. Stability studies of

aflatoxins using differential pulse stripping voltammetric (DPCSV) technique. Paper presented at SKAM-17, Kuantan, Pahang, 24 – 26th August 2004.

316 8

Yaacob, M.H., Mohd. Yusoff, A.R. and Ahamad, R. Stability studies of

aflatoxins using differential pulse stripping voltammetric (DPCSV) technique.

Malaysian J. Anal. Sci. In press. 9.

Yaacob, M.H., Mohd. Yusoff, A.R. and Ahamad, R. Voltammetric

determination of aflatoxins: Differential pulse voltammetric (PCSV) versus Squarewave stripping voltammetry (SWSV) techniques. Paper presented at KUSTEM 4 th Annual Seminar on Sustainibility Science and Management, Kuala Terengganu, 2nd – 3 rd May 2005. 10.

Yaacob, M.H., Mohd. Yusoff, A.R. , Ahamad, R. and Misni, M. Determination

of the aflatoxin B1 in groundnut samples by differential pulse cathodic stripping voltammetry (DPCSV) technique. Paper presented at Seminar Nasional Kimia II, Universiti Sumatera Utara, Medan, Indonesia. 14th April 2005. 11.

Yaacob, M.H., Mohd. Yusoff, A.R. Ahamad, R., and Misni, M. Development of

differential pulse cathodic stripping voltammetry (DPCSV) technique for the determination of aflatoxin B1 in groundnut samples. J Sains Kimia. 9(3): 31-36. 12.

Yaacob, M.H., Mohd. Yusoff, A.R. and Ahamad, R. Application of differential

pulse cathodic stripping voltammetry (DPCSV) technique in studying stability of aflatoxins. J Sains Kimia. 9(3): 64-70. 13.

Yaacob, M.H., Mohd. Yusoff, A.R. and Ahamad, R. Square-wave cathodic

stripping voltammetric (SWSV) technique for determination of aflatoxin B1 in groundnut samples. Paper presented at SKAM-18, JB, Johor. 12 – 14th September 2005. 14.

Yaacob, M.H., Mohd. Yusoff, A.R. and Ahamad, R. Square-wave cathodic

stripping voltammetric (SWSV) technique for determination of aflatoxin B1 in groundnut samples. Malaysian J. Anal. Sci. In press.

317

APPENDIX AM

ICP-MS results on analysis of BRB at pH 9.0

Figure AM-1

ICP-MS results on analysis of BRB at pH 9.0

Introduction

Experimental

Results & Discussion

Problem

Future Works

PRESENTED AT 1st ASSESMENT SEMINAR.

3/1/2007

1st ASSESSMENT REPORT

1

STRIPPING VOLTAMMETRIC STUDY FOR DETERMINATION OF AFLATOXIN COMPOUNDS

MOHAMAD HADZRI YAACOB (PS023001) SUPERVISOR: AP DR ABDUL RAHIM HJ MOHD YUSOFF CO-SUPERVISOR: PROF RAHMALAN AHAMAD

Introduction

Experimental

Results & Discussion

Problem

Future Works

TOPICS FOR DISCUSSION z

INTRODUCTION

AFLATOXINS

AFLATOXIN COMPOUNDS RESEARCH BACKGROUND OBJECTIVES SCOPE OF RESEARCH z z z z z z

z

EXPERIMENTAL RESULTS AND DISCUSSION CONCLUSION PROBLEMS SUGGESTIONS FOR FUTURE WORKS FLOW CHART OF FUTURE PLANNING ACKNOWLEDGEMENT

3/1/2007

1st ASSESSMENT REPORT

3

Introduction

Experimental

Results & Discussion

Problem

Future Works

INTRODUCTION

z

AFLATOXINS:

Mycotoxin group Produced by two fungi; Aspergillus flavus and Aspergillus parasiticus Types of aflatoxins: AFB1 AFB2 AFG1 AFG2 AFM1 AFM2

3/1/2007

Raw peanuts, peanuts products, wheat, maize, vegetable oils, dried fruites, barley, pear, cocoa, coffee, herbs, spices, wine, cottonseeds. Cow milk and its products, eggs and meat product 1st ASSESSMENT REPORT

4

Introduction

Experimental

Results & Discussion

Problem

Future Works

INTRODUCTION …cont

z AFLATOXINS: Occurs naturally in commodities used for animal and human Carcinogenic, teratogenic and mutagenic IARC (1988): Class I carcinogenic materials

Dangerous to health: will cause aflatoxicosis disease. Effect the economy of commodities export countries

3/1/2007

1st ASSESSMENT REPORT

5

Introduction

Experimental

Results & Discussion

Problem

Future Works

INTRODUCTION…cont

z AFLATOXINS Its growth depends on temperature and medium (acidity) Contamination of feed through infection and seed damaged by insects Enter into human body through consumption of contaminated food and skin absorption. Regulatory level in Malaysia: Raw peanuts Milk Others

3/1/2007

< 15 ppb < 0.5 ppb < 5 ppb

1st ASSESSMENT REPORT

6

Introduction

Experimental

Results & Discussion

Problem

Future Works

INTRODUCTION… cont

z

CHEMICAL STRUCTURES O

O

O

O

O O

O

O

O

O

O

O

AFB2

AFB1 O

O

O

O

O

O

O

O

O

O

O

O

O

AFG1 3/1/2007

O

AFG2 1st ASSESSMENT REPORT

7

Introduction

Experimental

Results & Discussion

Problem

Future Works

RESEARCH BACKGROUND z

Very dangerous to human health

z

Its actual amount must be determined by fast, accurate and relatively low cost technique / method.

z

Analytical level must be at ppb or ppt ( In Malaysia: HPLC with LOD:2.0 ppb and analysis time: 20 mins)

3/1/2007

1st ASSESSMENT REPORT

8

Introduction

Experimental

Results & Discussion

Problem

Future Works

RESEARCH BACKGROUND…cont Methods that have been used: z TLC ( 0.03 – 5.0 ppb) z HPLC (UVD, AD or FD) ( 0.014 – 7.0 ppb) z Enzyme linked immunosorbant assay (ELISA): ( 0.01 – 5.0 ppb) z Immunoaffinity chromatography (IAC): ( 0.2 – 2.0 ppb) z Micellar electrokinetic’s capillary chromatography (MECC): ( 0.02 – 0.09 ppb) z Differential pulse polagoraphy (DPP): ( 25 ppb) LOD of aflatoxins depends on the type of: aflatoxins, samples and extraction techniques

3/1/2007

1st ASSESSMENT REPORT

9

Introduction

Experimental

Results & Discussion

Problem

Future Works

RESEARCH BACKGROUND… cont z

Problems/disadvantages: TLC HPLC

Longer analysis time, need a lot of toxic organic solvents, high cost for instruments, and maintenance

ELISA

Reagents /kits are costly. Suitable for screen test.

RIA

3/1/2007

Longer analysis time, accuracy problem, aflatoxins absorption problem

Antibodies are costly, using dangerous radioisotopes, need special precaution for dealing with radioisotopes and its waste. It has certain half life. 1st ASSESSMENT REPORT

10

Introduction

Experimental

Results & Discussion

Problem

Future Works

RESEARCH BACKGROUND… cont

VOLTAMMETRY

Until now, no study has been performed using voltammetric techniques either cyclic voltammetry, cathodic/anodic stripping voltammetry or absorption stripping

Voltammetric technique has been chosen as an alternative method to be studied for determination of aflatoxins

3/1/2007

1st ASSESSMENT REPORT

11

Introduction

Experimental

Results & Discussion

Problem

Future Works

RESEARCH BACKGROUND …cont z

Voltammetric technique: sensitive, high accuracy, low cost and easy to use

3/1/2007

1st ASSESSMENT REPORT

12

Introduction

Experimental

Results & Discussion

Problem

Future Works

OBJECTIVES OF RESEARCH

(1)

(2)

TO STUDY ELECTROCHEMICAL PROPERTIES OF AFLATOXINS (AFB1, AFB2, AFG1 AND AFG2) USING VOLTAMMETRIC TECHNIQUE TOGETHER WITH CONTROL GROWTH MERCURY ELECTRODE (CGME) AS THE WORKING ELECTRODE

TO DEVELOPE A DETECTION METHOD FOR DETERMINATION OF AFLATOXINS IN FOOD SAMPLES WHICH IS SENSITIVE, SIMPLE, ACCURATE AND ECONOMIC TECHNIQUE.

CV

3/1/2007

DPSV

1st ASSESSMENT REPORT

13

Introduction

Experimental

Results & Discussion

Problem

Future Works

SCOPE OF STUDY

z

Using cyclic voltammetry (CV):-

9

Determine the type of aflatoxins reaction at mercury electrode when anodic or cathodic scanning is performed.

3/1/2007

1st ASSESSMENT REPORT

14

Introduction

Experimental

Results & Discussion

Problem

Future Works

SCOPE OF STUDY…cont

9

Identify the peak or peaks obtained either oxidation peak or reduction peak or both for developing a technique for analysis of aflatoxins.

3/1/2007

1st ASSESSMENT REPORT

15

Introduction

Experimental

Results & Discussion

Problem

Future Works

SCOPE OF STUDY…cont

z

Using voltammetric technique:

¾

study the effect of pH of electrolyte solution on reduction/oxidation aflatoxin peaks.

¾

determine the best electrolyte for the process of reduction / oxidation of aflatoxins at mercury electrode.

3/1/2007

1st ASSESSMENT REPORT

16

Introduction

Experimental

Results & Discussion

Problem

Future Works

SCOPE OF STUDY…cont

™ Parameters

optimisation to obtain maximum peak height and linear with addition of aflatoxins concentration. ™ Use optimum parameters for determination of AFB2, AFB1, AFG1 and AFG2

3/1/2007

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17

Introduction

Experimental

Results & Discussion

Problem

Future Works

PLANNING ACTIVITIES Activity 2002 M

J

J

O

S

O

N

D

1) Literature survey 2) Cyclic voltammetric study of AFB2 3) Stripping voltammetric study of AFB2 4) Parameters optimisation: 4a) using high concentration 4b) using low concentration

3/1/2007

1st ASSESSMENT REPORT

18

Introduction

Experimental

Results & Discussion

Problem

Future Works

PLANNING ACTIVITIES…cont Activity 2003 J

F

M

A

M

J

J

O

S

O

N

D

1) Determination of AFB1, AFB2, AFG1 and AFG2 1a) Prepare calibration curve 1b) Determine analytical parameters 2) Interference study 3) Complexing of aflatoxins study

3/1/2007

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19

Introduction

Experimental

Results & Discussion

Problem

Future Works

PLANNING ACTIVITIES…cont Activity 2004 J

F

M

A

M

J

J

O

S

O

N

D

1) Determination of mixing aflatoxins 2)Determination of aflatoxins in artificially contaminated samples 3)Determination of aflatoxins in real samples 4)Determination by HPLC 5) Study using different working electrodes 5) Comparison study 6) Thesis writting up

Activity 2005 1) Analysis of contaminated food samples 2) Thesis writting up

3/1/2007

1st ASSESSMENT REPORT

20

Introduction

Experimental

Results & Discussion

Problem

Future Works

STUDY FLOW CHART AFLATOXIN COMPOUNDS

AFB1

AFB2

Cyclic voltammetric, anodic/cathodic

Direct measurement

AFG1

AFG2

Differential pulse stripping Voltammetric, anodic / cathodic

Complexing with metals (Zn, Pb, Cu) or Organic materials (Cystein)

Modified electrode / Screen printed electrode

Method optimisation

3/1/2007

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21

Introduction

Experimental

Results & Discussion

Problem

Future Works

STUDY FLOW CHART…cont

Optimise;E acc, tacc, Ei, Ef, scan rate, pulse amplitude

Method validation

Limit of detection

By experiment

3/1/2007

By graph

Linearity

Precision

Intra-day

1st ASSESSMENT REPORT

Accuracy

Inter-day

22

Introduction

Experimental

Results & Discussion

Problem

Future Works

STUDY FLOW CHART…cont

Method testing Mixing aflatoxins std (AFB1,AFB2,AFG1,AFG2)

Real samples

Artificially contaminated samples

Compare the results with that obtained by HPLC

Method is tested at different laboratory / analyser

Method is accepted

3/1/2007

1st ASSESSMENT REPORT

23

Introduction

Experimental

Results & Discussion

Problem

Future Works

EXPERIMENTAL: INSTRUMENTS AND REAGENTS

z

INSTRUMENTS: BAS CGME (consist of Ag/AgCl as RE, Pt wire as AE and HMDE as WE) interface with BAS C50W Voltammetric Analyser

z

Electrolyte solutions: BRB 0.04M at various pH (2.0,3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0,10.0,11.0, 12.0 dan 13.0). 0.1M HCl for pH 1.0

z

Standard solutions: AFB1, AFB2, AFG1 dan AFG2 10 ppm (10ml) – SIGMA

z

Techniques: CV dan DPCSV

3/1/2007

1st ASSESSMENT REPORT

24

Introduction

Experimental

Results & Discussion

Problem

Future Works

EXPERIMENTAL FLOW CHART

10 ML BRB SOLUTION

NITROGEN GAS IS FLOWED IN AFLATOXINS SOLUTION, REDISSOLVED IN BRB SOLUTION

SPIKE INTO 20 ML VOLTAMMETRIC CELL

SOLUTION IS DEGASSED

BLANK SOLUTION BLANK VOLTAMMOGRAM

3/1/2007

SOLUTION IS SCANNED OBTAINED VOLTAMMOGRAM

1st ASSESSMENT REPORT

BLANK SOLUTION + AFLATOXIN SAMPEL VOLTAMMOGRAM 25

Introduction

Experimental

Results & Discussion

Problem

Future Works

RESULTS AND DISCUSSION

z

CV STUDY ( [AFB2] = 1.3 x 10-6M in BRB pH 9.0) Ip for AFB2 = 30 nA Ep = -1315 mV

No anodic peak

Irreversible reaction Scan rate = 200mV/s 1 = cathodic direction 2 = anodic direction

3/1/2007

1st ASSESSMENT REPORT

26

Introduction

Experimental

Results & Discussion

Problem

Future Works

RESULTS AND DISCUSSION…cont

z

CV : to confirm the cathodic peak

z

[AFB2] = 1.3 x 10-6M in BRB pH = 9.0

Ip AFB2 = 35 nA Ep = -1311 mV

No anodic peak

Irreversible reaction 1. Anodic direction 2. Cathodic direction

3/1/2007

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27

Introduction

Experimental

Results & Discussion

Problem

Future Works

RESULTS AND DISCUSSION…cont

z

CV : to confirm the cathodic peak

z

By [AFB2] standard addition and cathodic scanning in BRB pH 9.0 (a) 1.3 uM 30nA, -1315 mV (b) 2.0 uM 47 nA, -1313 mV (c) 2.7 uM 67 nA, -1306 mV (d) 3.4 uM 79 nA, -1303 mV 100 Ip (nA)

80 60 40

y = 26.541x - 3.035

20

R2 = 0.9895

0 0

1

2

3

4

[AFB2] X 10-6M

3/1/2007

1st ASSESSMENT REPORT

28

Introduction

Experimental

Results & Discussion

Problem

Future Works

RESULTS AND DISCUSSION…cont

Repeatitive cathodic scanning using single mercury drop, 1.3 x 10-6M AFB2 in BRB pH = 9.0

No of Ep cycle (-mV)

60 50 i p c a th o d i c (n A )

z

40 30 20 10 0 1

3

5 no of cycle

7

9

1 3 5 7 9

1314 1310 1309 1307 1304

Adsorption process at mercury electrode O

O

AFB2 O

Diffusion current is influenced by adsorption process at mercury electrode

O

O O

3/1/2007

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29

Experimental

Introduction

Results & Discussion

Problem

Future Works

RESULTS AND DISCUSSION…cont

z

Effect of scan rate (V) on Ip and Ep of AFB2 (1) Log Ep vs log V 1.9 1.7

lo g E p

1.5

Linear with m >0.5:

1.3 1.1 0.9

y = 0.5097x + 0.4115 R2 = 0.995

0.7 0.5 1.2

1.4

1.6

1.8

2 log V

3/1/2007

2.2

2.4

2.6

2.8

ÎDiffusion current produced by reduction process of aflatoxins which is influenced by adsorption process at mercury electrode.

1st ASSESSMENT REPORT

30

Experimental

Introduction

Results & Discussion

Problem

Future Works

RESULTS AND DISCUSSION…cont

z

Effect of scan rate (V) on Ip and Ep of AFB2 (2) Ep vs log V

1340

E p (-m V )

1330

Linear :

1320 1310

Ep AFB2 changing with Increasing of V.

1300 1290

y = 40.065x + 1223.3 R2 = 0.9936

1280 1270 1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

ÎIrreversible reaction

log V

3/1/2007

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31

Experimental

Introduction

Results & Discussion

Problem

Future Works

RESULTS AND DISCUSSION…cont

z

Effect of scan rate (V) on Ip of AFB2 (3) Ip vs V 70

Linear : two different regions due to different scan rate (1) 100 – 500 mV/s (fast) : m = 0.076 R = 0.9917

60

(2) 20 – 100 mV/s (slow): m = 0.264 R = 0.9979

Ip (n A )

50 40 30 20 10 0 0

50

100 150 200 250

300 350 400 450 500 550

V (mv/s)

3/1/2007

Linear for both regions: AFB2 is adsorped at mercury electrode. Î Reduction and adsorption processes at electrode are influenced by the rate of reaction which prefers the slow reaction.

1st ASSESSMENT REPORT

32

Introduction

Experimental

Results & Discussion

Problem

Future Works

RESULTS AND DISCUSSION…cont

AFB2 reduction reaction: : Irreversible

z

1. CV produces only cathodic peak. 2. Graph Ep vs log V: linear

Cathodic Stripping Volltammetric is selected : Diffusion current is 1. graph log Ep vs log V: linear, influenced by m = >0.5 adsorption 2. graph Ip vs V: linear with process with higher m at slow V compare prefer the slow to the m at fast V 3. I increases with repeatitive reaction cathodic scanning. p

3/1/2007

1st ASSESSMENT REPORT

33

Introduction

Experimental

Results & Discussion

Problem

Future Works

RESULTS AND DISCUSSION…cont

z

Differential Pulse Cathodic Stripping Voltammetric (DPCSV) technique

(1) -1256mV, 5nA

(1)Scan [AFB2] = 1.0 nM Ei = 0, Ef = -1500 mV, Eacc =0 mV T acc = 0, v = 50 mv/s, pulse amplitude: 100 mV (in BRB pH =9.0) (2)Scan [AFB2] = 1.0 nM Ei = 0, Ef = -1500 mV, Eacc =0 mV T acc = 30s, v = 50 mv/s, pulse amplitude: 100 mV (In BRB pH = 9.0)

-1244mV, 25nA

(2)

Accumulation time greatly effect on peak height (Ip) of AFB2 3/1/2007

1st ASSESSMENT REPORT

34

Introduction

Experimental

Results & Discussion

Problem

Future Works

RESULTS AND DISCUSSION…cont z

Study effect of pH of supporting electrolyte ( BRB ) Aims: a) To study the pH of BRB that gives maximum Ip of AFB2 without giving any extra peak. b) To study either hidrogen ion is greatly involved in the reduction reaction of AFB2 or not. [AFB2] = 2.0 uM, BRB with pH = 2.0 to 13.0, use 0.1M HCl for pH = 1.0 Method: DPCSV Parameters:

Ei = 0, Ef = -1500 mV, Eacc = 0, t acc = 15 s, v = 50 mV/s 3/1/2007

1st ASSESSMENT REPORT

35

Experimental

Introduction

Results & Discussion

Problem

Future Works

RESULTS AND DISCUSSION…cont

Ip (nA)

[AFB2] = 2.0 uM, BRB with pH = 2.0 to 13.0, 0.1 M HCl for pH=1.0 Method: DPCSV Parameters: Ei = 0, Ef = -1500 mV, Eacc = 0, t acc = 15s, v = 50 mV/s pH =9.0, optimum pH Î Ip = 48 nA, Ep = -1192 mV

60 50 40 30 20 10 0 4

6

8

10

12

pH < 5.0, > 12.0 : no AFB2 peak pH: 5.0 – 9.0: Ip increased pH: 9.0 – 12.0: Ip decreased

pH

BRb pH 9.0 is selected 3/1/2007

The reaction is effected by pH of supporting electrolyte. =Î proton ion is greatly involve in reduction reaction of AFB2.

1st ASSESSMENT REPORT

36

Introduction

Experimental

Results & Discussion

Problem

Future Works

RESULTS AND DISCUSSION…cont

z

Voltammograms of AFB2 with changing pH

pH = 4.0 No peak

3/1/2007

1st ASSESSMENT REPORT

pH = 7.0 ( - 1168 mV, 42 nA)

37

Introduction

Experimental

Results & Discussion

Problem

Future Works

RESULTS AND DISCUSSION…cont z

Voltammograms of AFB2 with changing pH

Interfered by blank ( BRb pH = 9.0 ) pH = 9.0 ( -1192 mV, 48 nA)

3/1/2007

pH = 11.0 (-1193 mV, 32 nA)

1st ASSESSMENT REPORT

38

Experimental

Introduction

Results & Discussion

Problem

Future Works

RESULTS AND DISCUSSION…cont

z

Effect of pH of BRB on Ep of AFB2 pH: 5.0 – 8.0: linear: R = 0.9777

E p (-m V )

1250 1200

pH: 9.0 – 12.0: nearly constant

1150 1100 4

6

8

10 pH

12

14

Ep shifted towards more negative direction =Î At or before optimum pH (9.0), reaction is effected by H+ ion

3/1/2007

1st ASSESSMENT REPORT

39

Introduction

Experimental

Results & Discussion

Problem

Future Works

RESULTS AND DISCUSSION…cont z Parameters

optimisation: z Scan rate (v) z Accumulation potential ( Eacc) z Accumulation time (tacc) z Scanning potential window( Ei and Ef) z Pulse amplitude

3/1/2007

1st ASSESSMENT REPORT

40

Introduction

Experimental

Results & Discussion

Problem

Future Works

RESULTS AND DISCUSSION…cont z Parameters

optimisation steps

Using two different concentrations of AFB2 (1) High:

(2) Low:

[AFB2] = 2.0 uM

[AFB2] = 0.06 uM

Optimum parameters for determination of AFB2 using DPCSV technique

3/1/2007

1st ASSESSMENT REPORT

41

Introduction

Experimental

Results & Discussion

Problem

Future Works

RESULTS AND DISCUSSION…cont Initial parameters: Ei = 0,Ef = -1500 mV,Eacc = 0, tacc = 15 s, V = 20 mv/s and P.A = 100 mV. [AFB2] = 2.0 uM Effect of Hg oxidation -1240 mV , 29 nA

H2 overpotential Interfered by blank ( BRb pH 9.0 )

3/1/2007

1st ASSESSMENT REPORT

42

Experimental

Introduction

Results & Discussion

Problem

Future Works

RESULTS AND DISCUSSION…cont Effect of scan rate (v) Ip (nA)

z

Optimum V : 40 mv/s Ip = 36 nA, Ep = -1256 mV Initial Ip : 29 nA

50 40 30 20 10 0 0

20

40

60

80

100

120

Scan rate (mV/s)

1) At higher v, Ip decreased due to uncomplete reaction.

Ep (-mV)

1320 1290 1260 1230 0

30

60

90

120

2) Higher v caused Ep linearly ( R2 = 0.9868) shifted to more negative direction.

Scan rate (mV/s)

V = 40 mV/s

3/1/2007

reduction reaction of AFB2 at mercury electrode prefered slow reaction

1st ASSESSMENT REPORT

43

Experimental

Introduction

Results & Discussion

Problem

Future Works

RESULTS AND DISCUSSION…cont z

Effect of accumulation time (tacc) Optimum tacc : 40s Ip = 45 nA, Ep = -1256 mV Initial Ip : 36 nA

Ip (n A )

60 40 20 0 0

20

40

60

Accumulation time (s)

3/1/2007

Tacc > 40s: Ip decreased due to the formation of saturated layer at mercury electrode. 2. Amount of analyte (AFB2) in the bulk solution is lower due to the adsorption process at mercury electrode

1st ASSESSMENT REPORT

44

Experimental

Introduction

Results & Discussion

Problem

Future Works

RESULTS AND DISCUSSION…cont z

Effect of accumulation potential(Eacc) Optimum Eacc: -800 mv Ip = 51nA, Ep = -1248 mV Initial Ip : 45 nA

Ip (nA )

60 40 20 0 0

400

800

1200

1600

Accumulation potential (-mV)

When: Eacc = Ei ( 0 mV): Ip lower Eacc = Ef ( -1500 mV): no peak was observed

At Eacc = -800 mV, AFB2 maximum adsorped at mercury electrode

3/1/2007

1st ASSESSMENT REPORT

45

Introduction

Experimental

Results & Discussion

Problem

Future Works

RESULTS AND DISCUSSION…cont

Ip (n A )

z

Effect of initial potential 70 60 50 40 30 20 10 0

Optimum Ei: -1000 mv Ip = 53 nA, Ep = -1256 mV Initial Ip : 51 nA

Scanning from Ei =-1000 mV increasing Ip of AFB2 because it can minimised the effect of blank (which give a peak around -950mV) 0

300

600 Ei (-mV)

900

1200

Ei at -1000 mV will save scanning time

Ei = -1000 mV and Ef = -1500 mV is selected as an opimum potential window. 3/1/2007

1st ASSESSMENT REPORT

46

Introduction

Experimental

Results & Discussion

Problem

Future Works

RESULTS AND DISCUSSION…cont z

Effect of pulse amplitude (P.A) Optimum P.A: 100 mv Ip = 53 nA, Ep = -1256 mV Initial Ip : 53 nA (same P.A)

Ip (nA )

80 60

Not selected eventhough Ip increased because:

40 20 0 0

30

60

90

120

Pulse amplitude (mV)

150

a) Very small increment of Ip, only 1nA ( 54nA) b) Peak broader and produces higher noise level

P.A =100 mV is selected as the optimum P.A 3/1/2007

1st ASSESSMENT REPORT

47

Introduction

Experimental

Results & Discussion

Problem

Future Works

RESULTS AND DISCUSSION…cont Initial parameters 2.0 µM AFB2 in BRB pH 9.0

Ei = 0, Ef = -1500 mV, Eacc = 0, tacc = 15 s, scan rate = 20 mv/s and P.A = 100 mV Ip = 29 nA, Ep = -1240 mV

Ei = -1000 mV, Ef = -1500 mV, Eacc =-800 mV, tacc = 40 s, Scan rate = 40 mV/s and P.A = 100 mV,

Optimum parameters

Ip = 53 nA (↑83%) , Ep = -1208 mV

3/1/2007

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48

Introduction

Experimental

Results & Discussion

Problem

Future Works

RESULTS AND DISCUSSION…cont Using lower concentration of AFB2

0.06µM AFB2 In BRB pH 9.0

Reoptimisation steps

Using previous optimum parameters Ei = -1000 mV, Ef = -1500 mV, Eacc =-800 mV, tacc = 40 s, s.rate = 40 mV/s and P.A = 100 mV 13.9nA, -1232 mV

Obtained new optimum parameters Ei = -1000 mV, Ef = -1400 mV, Eacc =-600 mV, tacc = 80 s, s.rate = 50 mV/s and P.A = 80 mV

38.18 nA, ( ↑200%), -1220mV

( Final optimum parameters) 3/1/2007

1st ASSESSMENT REPORT

49

Introduction

Experimental

Results & Discussion

Problem

Future Works

RESULTS AND DISCUSSION…cont Before optimised: Ei = -1000 mV, Ef = -1500 mV, Eacc =-800 mV, tacc = 40 s, s.rate = 40 mV/s and P.A = 100 mV

-1232 mV, 13.90 nA

-1220 mV, 38.18 nA After optimised: Ei = -1000 mV, Ef = -1400 mV, Eacc =-600 mV, tacc = 80 s, s.rate = 50 mV/s and P.A = 80 mV

Optimum parameters for determination of AFB2 [AFB2] = 0.06 µM 3/1/2007

1st ASSESSMENT REPORT

50

Introduction

Experimental

Results & Discussion

Problem

Future Works

RESULTS AND DISCUSSION…cont z

Preparation of calibration curve ( AFB2 in BRB pH 9.0) 200

ip (nA)

150 100 y = 5.55x + 3.6435

50

R2 = 0.9978

0 0

10

20

30

40

[AFB2] X10-8M

Ei = -1000 mV, Ef = -1400 mV, Eacc =-600 mV, tacc = 80 s, s.rate = 50 mV/s and P.A = 80 mV

Linear range: 0.02 – 0.32 µM LOD: 0.0159 µM ( 5.0 ppb) Sensitivity: 0.555 µA / µM

Ep AFB2 (0.1 uM) = -1230 mV 3/1/2007

1st ASSESSMENT REPORT

51

Introduction

Experimental

Results & Discussion

Problem

Future Works

RESULTS AND DISCUSSION…cont z From

calibration curve:

1) Precision : Determine the AFB2 with concentrations of (1) 0.06 µM and (2) 0.10 µM. Calculate relative standard deviation (RSD) for each determination. ( repeatibility ) ( RSD < 5%) 2) Accuracy: Determine the recovery by scanning a known amount of AFB2 and calculate the actual amount of AFB2 using regression equation which was obtained from the calibration curve. The obtained value is compared with amount of spiked AFB2. (recovery) 3/1/2007

1st ASSESSMENT REPORT

52

Introduction

Experimental

Results & Discussion

Problem

Future Works

RESULTS AND DISCUSSION…cont

z

Precision:

(2)

(1)

[AFB2] =0.06 µM, n = 8, RSD = 2.80%

[AFB2] =0.20 µM, n = 8, RSD = 2.53%

RSD = < 5%: Proposed method is high precision

3/1/2007

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Introduction

Experimental

Results & Discussion

Problem

Future Works

RESULTS AND DISCUSSION…cont z

Precision: (inter-day) Day 2

[AFB2] = 0.1 uM Day 1

Day 3 Day 1: 72.70 +/- 0.521

( 0.72% ) , n = 8

Day 2: 72.22 +/- 0.533

( 0.74% ) ,,

Day 3: 72.91 +/- 0.706

( 0.97% ) ,,

Average: 72.61 +/- 0.587

( 0.81% ) ,,

High precision for intra and inter day measurements 3/1/2007

1st ASSESSMENT REPORT

54

Introduction

Experimental

Results & Discussion

Problem

Future Works

RESULTS AND DISCUSSION…cont z

Accuracy (recovery) n = 3 No of experiment

1

2

3/1/2007

Amount added (x 10-8M)

Peak current (nA)

Amount found (x 10-8M)

10.0

51.70 52.77 51.03

8.66 8.84 8.55

86.83 +/- 1.46 ( 1.68%)

25.0

134.4 133.3 135.6

23.56 23.36 23.77

94.26 +/- 0.81 (0.86%)

1st ASSESSMENT REPORT

Recovery (%) (RSD)

55

Introduction

Experimental

Results & Discussion

Problem

Future Works

RESULTS AND DISCUSSION…cont z

Application to determine AFG2, AFB1 and AFG1 AFG2, AFB1 and AFG1 : CV Î In BRB pH 9.0, all aflatoxins are reduced at mercury electrode produced single cathodic peak and the reactions are irreversible. Diffusion currents are effected by adsorption process at electrode surface

=AFB2 DPCSV Î used the same optimum parameters as used for AFB2 to determine AFG2, AFB1 dan AFG1

3/1/2007

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56

Introduction

Experimental

Results & Discussion

Problem

Future Works

RESULTS AND DISCUSSION…cont Voltammograms of AFB1 (A), AFB2 (B), AFG1 (C) and AFG2 (D) obtained from individual measurement at concentration of 0.1 uM in BRB pH 9.0

Ei =-1000 mV ( except for AFG1 = -950 mV), Ef = -1400 mV, Eacc = -600 mV, t acc = 80 s, v = 50 mV/s and p.amplitude = 80 mV (A) AFB1 = -1220 mV ( 62.57 nA ), (B) AFB2 = -1230 mV ( 63.79 nA ) (C) AFG1 = -1150 mV ( 62.29 nA ), (D) AFG2 = -1170 mV ( 63.14 nA ) 3/1/2007

1st ASSESSMENT REPORT

57

Introduction

Experimental

Results & Discussion

Problem

Future Works

RESULTS AND DISCUSSION…cont z

Saturation effect at electrode surface

AFG2:

200

300

150

200

Ip (nA)

Ip (nA)

250 150 100

100 y = 6.3464x + 9.8329 R2 = 0.999

50

50 0

0 0

10

20

30

40 50

60

70

80

90

0

5

10

15

20

25

30

35

[AFB2]X10-8M

[AFG2] / X10-8M

y = 6.346x + 9.8329 ( R =0.9990) Linear range: 0.024 uM – 0.288 uM LOD: 0.0160 uM (5.28 ppb) Sensitivity: 0.635 uA/uM Ei = -1000 mV, Ef = -1400 mV, Eacc =-600 mV, tacc = 80 s, s.rate = 50 mV/s and P.A = 80 mV 3/1/2007

Ep= -1170mV

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Introduction

Experimental

Results & Discussion

Problem

Future Works

RESULTS AND DISCUSSION…cont z

AFB1

250 200 Ip (nA)

300

Ip (n A )

250 200

150 100

150

50

100

0

50

y = 4.4296x + 3.0034 2

R = 0.9972

0

0 0

20

30

40

50

[AFB1]X10-8M

10 20 30 40 50 60 70 80 90 100 [AFB1]X10-8M

10

Y = 4.4296x + 3.0034 ( R =0.9972) Linear range : 0.0321 uM – 0.5778 uM LOD: 0.0161 uM ( 5.00 ppb) Sensitivity: 0.443 uA/uM

Parameters for AFB1 = AFG2 = AFB2 Ep = -1220 mV 3/1/2007

1st ASSESSMENT REPORT

59

Experimental

Introduction

Results & Discussion

Problem

Future Works

RESULTS AND DISCUSSION…cont AFG1 200 175 150 125 100 75 50 25 0

Ip (n A)

Ip (nA)

z

0

10

20

30

40

50

60

160 140 120 100 80 60 40 20 0

y = 5.5958x + 3.7725 R2 = 0.998

0

5

10

15

20

25

30

[AFG1]X10-8M

[AFG1] X 10-8M

y = 5.5958x + 3.7725 Linear range: 0.02 uM – 0.260 uM LOD: 0.0150 uM ( 4.92 ppb) Sensitivity: 0.560 uA/uM Ei = -950 mV, Ef = -1400 mV, Eacc =-600 mV, tacc = 80 s, s.rate = 50 mV/s and P.A = 80 mV

3/1/2007

1st ASSESSMENT REPORT

Ep= -1150mV

60

Introduction

Experimental

Results & Discussion

Problem

Future Works

RESULTS AND DISCUSSION…cont Voltammograms of AFG1 Ip (n A)

z

160 140 120 100 80 60 40 20 0

y = 5.5958x + 3.7725 R2 = 0.998

0

5

10

15

20

25

30

[AFG1]X10-8M

Ei = -950 mV, Ef = -1400 mV, Eacc =-600 mV, tacc = 80 s, s.rate = 50 mV/s and P.A = 80 mV

y = 5.5958x + 3.7725 R = 0.9980

Ep AFG1 (0.1 uM) = -1150 mV 3/1/2007

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Experimental

Introduction

Results & Discussion

Problem

Future Works

RESULTS AND DISCUSSION…cont z Aflatoxin

AFB1, AFG1 dan * Y = mx + c AFG2: Sensitivity E (mV)AFB2, p

(y = nA, x = x 10-8M,

LOD

Linear range

( x 10-8M)/

(x 10-8M) /

ppb

ppb

0.443

1.606/ 5.00

3.00 – 45.00/ 10 - 150

0.9972

(uA/uM)

c = nA) Y = 4.4296x + 3.0034

R2

AFB1

-1220

AFB2

-1230

Y =5.5500x + 3.6435

0.555

1.580/ 5.00

2.00 – 32.00/ 6 - 100

0.9976

AFG1

-1150

Y = 5.5958x + 3.7725

0.560

1.500/ 4.92

2.00 – 26.00/ 6 - 90

0.9980

AFG2

-1170

Y = 6.3464x + 9.8329

0.635

1.600/ 5.28

2.00 – 29.00/ 6 -96

0.9990

* [aflatoxins] = 0.1 uM 3/1/2007

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Introduction

Experimental

Results & Discussion

Problem

Future Works

CONCLUSION z From

the CV study, AFB1, AFB2, AFG1 and AFG2 have reduced at a mercury electrode which produced diffusion current that controlled by adsorption process at electrode surface. The reduction reaction was totally irreversible and prefered the slow reaction.

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Introduction

Experimental

Results & Discussion

Problem

Future Works

CONCLUSION…cont z From

the DPCSV, in BRB pH 9.0, AFB1, AFB2, AFG1, AFG2 have reduced at peak potentials of -1220 mV, -1230 mV, 1150 mV and -1170 mV ( versus Ag/AgCl) respectively. z The proposed method provides high precision and accuracy for determination of AFB2. 3/1/2007

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Introduction

Experimental

Results & Discussion

Problem

Future Works

PROBLEMS 1. Blank BRB pH 9.0 gives a peak ( around -950 mV to -1100 mV) with unconsistent Ip (15 nA – 40 nA)

(a)

Ei = 0, Ef = -1500 mv Eacc = 0 tacc = 20 s

3/1/2007

(b)

Ei = 0, Ef = -1500 mv Eacc = 0 tacc = 20 s

1st ASSESSMENT REPORT

(c)

Ei = -1000 mv, Ef = -1400 mv Eacc = -600 mv tacc = 80 s

65

Introduction

Experimental

Results & Discussion

Problem

Future Works

PROBLEMS…cont 2. Initial analysis of mixing aflatoxins sample: : AFB2 + AFG2 Î 1 overlap peak : AFB1+ AFB2 Î 1 overlap peak 3. Initial analysis of real sample (uncontaminated green nut powder ): The sample supress AFB2 standard peak almost 50%. ( recovery: 50%)

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Introduction

Experimental

Results & Discussion

Problem

Future Works

FUTURE WORKS z Further

study on suitable technique to reduce LOD / enhance sensitivity. z Analysis of accuracy ( recovery) at interday for AFB2. z Analysis of precision and accuracy at intra-day and inter-day for AFG2, AFB1 dan AFG1

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Introduction

Experimental

Results & Discussion

Problem

Future Works

FUTURE WORKS…cont

ÎComplexing of aflatoxin for the selectivity of analysis. ÎStudy interference effect; 1. Analysis the mixing of aflatoxins sample. 2. Standard aflatoxin solution will be mixed with other ketone compounds. 3/1/2007

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Introduction

Experimental

Results & Discussion

Problem

Future Works

FUTURE WORKS…cont z

Study the stability of aflatoxins ( shelf life study) and the effect of exposure time to the aflatoxins peak current ( aflatoxins will be left in voltammetric cell subject to the laboratory conditions for 8 hrs and measurement will be carried out for every hour)

z

Study other type of working electrodes such as carbon paste and screen printed electrodes.

z

Robustness test of the proposed method.

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Introduction

Experimental

Results & Discussion

Problem

Future Works

FUTURE WORKS…cont z Analysis

an artificially and real contaminated samples.

z Compare

the results with that obtained using accepted technique such as HPLC-FD.

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Introduction

Experimental

Results & Discussion

Problem

Future Works

FUTURE WORKS…cont Activity 2003 J

F

M

A

M

J

J

O

S

O

N

D

1) Determination of AFB1, AFB2, AFG1 and AFG2 1a) Prepare calibration curve

finished

1b) Determine analytical parameters 2) Interference study 3) Complexing of aflatoxins study

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Introduction

Experimental

Results & Discussion

Problem

Future Works

FUTURE WORKS…cont Activity 2004 J

F

M

A

M

J

J

O

S

O

N

D

1) Determination of mixing aflatoxins 2)Determination of aflatoxins in artificially contaminated samples 3)Determination of aflatoxins in real samples 4)Determination by HPLC 5) Study using different working electrodes 5) Comparison study 6) Thesis writting up

Activity 2005 1) Analysis of contaminated food samples 2) Thesis writting up

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Introduction

Experimental

Results & Discussion

Problem

Future Works

WHAT EXPERTS SAID ABOUT VOLTAMMETRY? Prof Barek (2001): “Polarographic and voltammetric methods can play a useful role in the analysis as an alternative to chromatographic and spectrophotometric techniques, however, their relationship to other methods is complimentry rather than competitive” Prof Wang (1999): “Stripping analysis to the 21th century: faster, smaller, cheaper, simpler and better”. Hopefully this will come true!!!

Jaroslav Heyrovsky (1890-1967) 3/1/2007

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Introduction

Experimental

Results & Discussion

Problem

Future Works

ACKNOWLEDGEMENT SUPERVISOR: AP DR ABDUL RAHIM MOHD YUSOFF CO-SUPERVISORS: 1. PROF RAHMALAN AHAMAD 2. DR ABD. RAHIM YAACOB STAFF OF CHEMISTRY DEPT, UTM CHEM. DEPT, MOSTE ( EN.RODWAN ) ELECTROANALYSIS GROUP, CHEM. DEPT, UTM USM ( STUDY LEAVE UNDER ASHES)

3/1/2007

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74

Introduction

Experimental

Results & Discussion

Problem

Future Works

THANK YOU Wassalam Mohd Hadzri, Hadzri, PPSains Kesihatan 20 Ogos 2003/ 22 Rejab 1424H

3/1/2007

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75

Introduction

3/1/2007

Experimental

Results & Discussion

Problem

Future Works

1st ASSESSMENT REPORT

76

Introduction

3/1/2007

Experimental

Results & Discussion

Problem

Future Works

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77

Introduction

Experimental

Results & Discussion

Problem

Future Works

LOD DETERMINATION

LOD = limit of detection LOD of an analyte is the concentration which gives an instrumental signal (y) significantly different from the blank or background signal ( Miller J. C. and Miller J. N. 1993)

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Introduction

Experimental

Results & Discussion

Problem

Future Works

LOD DETERMINATION…cont

z By

Barek’s and Miller’s methods z Barek’s method: a) experiment b) graph 1a) Experiment: By standard addition of lower concentration of analyte until obtaining the sample response that is significantly difference from blank signal 3/1/2007

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79

Introduction

Experimental

Results & Discussion

Problem

Future Works

LOD DETERMINATION…cont

1b)By graph: The limit of determination was calculated as the tenfold standard deviation from seven analyte determinations at the concentration corresponding to the lowest point on the appropriate calibration straight line z Miller’s method: By graph; 3SD/b z

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Introduction

Experimental

Results & Discussion

Problem

Future Works

LOD DETERMINATION…cont

By Barek’s Method

By Miller’s Method

Aflatoxin Experiment

Graph

Graph; 3SD/b

x 10-8 M

ppb

x 10-8 M

ppb

x 10-8 M

ppb

AFB1

1.606

5.00

2.160

6.70

45.36

141.5

AFB2

1.580

5.00

1.837

5.77

28.59

89.0

AFG1

1.500

4.92

2.050

6.72

23.88

78.33

AFG2

1.600

5.28

1.345

4.44

27.47

90.67

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Introduction

Experimental

Results & Discussion

Problem

Future Works

BLANK PEAK PROBLEM

z Attempt

to illiminate; 1. Used dionised water from different lab; (Analytical, biological, FKKKSA, Biotechnology) and R.O water from personal used….failed

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Introduction

Experimental

Results & Discussion

Problem

Future Works

BLANK PEAK PROBLEM…cont

2. Add 1.0 M HCl … failed 3. Add 1.0 M EDTA…failed 4. Used different voltammetry model (Metrohm) peak still existed

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83

Introduction

Experimental

Results & Discussion

Problem

Future Works

STABILITY STUDY OF AFLATOXINS

z

AFB1, hour to hour stability study Stability study of AFB1 in BRb pH=9.0 60

Ip = 58.53 +/- 0.62 nA

( 1.06%)

[AFB1] = 10 x 10-8M

Ip (n A )

58 56 54 52 50 0

1

2

3

4

5

6

7

8

Standing time (hrs)

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84

Experimental

Introduction

Results & Discussion

Problem

Future Works

STABILITY STUDY OF AFLATOXINS…cont

z

AFB2, hour to hour stability study

Ip = 72.74 +/- 0.94 ( 1.29 % )

Stability study of AFB2 in BRB pH 9.0 73.5

[AFB2] =12 x 10-8M

73 Ip (nA)

72.5 72 71.5 71 70.5 70 1

2

3

4

5

6

7

8

9

Standing time (hrs)

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85

Introduction

Experimental

Results & Discussion

Problem

Future Works

CV OF AFB1

z

Cyclic voltammogram of AFB1 in BRB pH 9.0, scan rate 200 mV/s Ip = 50 nA, Ep = -1305 mV

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86

Introduction

Experimental

Results & Discussion

Problem

Future Works

CV OF AFG1

z

Cyclic voltammogram of AFG1 in BRB pH 9.0, scan rate 200 mV/s Ip = 48 nA, Ep = -1245 mV

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Introduction

Experimental

Results & Discussion

Problem

Future Works

CV OF AFG2

z

Cyclic voltammogram of AFG2 in BRB pH 9.0, scan rate 200 mV/s Ip = 52 nA, Ep= -1254 mV

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Introduction

Experimental

Results & Discussion

Problem

Future Works

VOLTAMMOGRAMS OF AFB2

z

DPSV voltammograms of AFB2 in BRB pH 9.0 (blank), scan rate 50 mV/s

AFB2 ( 2.0 uM ) Blank, BRB pH 9.0

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Introduction

Experimental

Results & Discussion

Problem

Future Works

VOLTAMMOGRAMS OF STD AFB2 AND REAL SAMPLE

AFB2, 0.07 uM ( 35.83 nA, -1240 mV AFB2 + REAL SAMPLE ( 16.90 nA, -1245 mV)

BLANK

USED OPTIMISED PARAMETERS FOR AFB2 REAL SAMPLE: EXTRACT FROM GREEN NUT POWDER 3/1/2007

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90

Introduction

Experimental

Results & Discussion

Problem

Future Works

VOLTAMMOGRAM OF MIXED AFLATOXINS

z

AFG2 + AFB2

0.1 uM AFG2 ( before mix) ( 64 nA, -1170 mV) + 0.1 uM AFB2 ( 61.5 nA, -1170mV) + 0.2 uM AFB2 (60.8 nA, -1200 mV) 3/1/2007

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Introduction

Experimental

Results & Discussion

Problem

Future Works

VOLTAMMOGRAM OF MIXED AFLATOXINS…cont

z

AFG2 + AFB2

+ 0.3 uM AFB2 ( 76 nA, -1210)

3/1/2007

+ 0.4 uM AFB2 ( 79 nA, -1210 mV)

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Introduction

Experimental

Results & Discussion

Problem

Future Works

VOLTAMMOGRAM OF MIXED AFLATOXINS…cont

0.1 uM AFG2 ( before mixed): -1170 mV, 64.0 nA

+ 0.05 uM AFB2: -1170 mV, 62.7 nA + 0.10 uM AFB2: -1170 mV, 61.5 nA + 0.15 uM AFB2: -1170 mV, 58.2 nA + 0.20 uM AFB2: -1200 mV, 60.8 nA + 0.25 uM AFB2: -1210 mV, 69.9 nA + 0.30 uM AFB2: -1210 mV, 76.0 nA + 0.35 uM AFB2: -1210 mV, 79.0 nA + 0.40 uM AFB2: -1210 mV, 79.0 nA 3/1/2007

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Introduction

3/1/2007

Experimental

Results & Discussion

Problem

Future Works

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94

WILL BE PRESENTED FOR PhD VIVA PRESENTATION

3/1/2007

PhD viva presentation

STRIPPING VOLTAMMETRIC METHOD TO DETERMINE AFLATOXIN COMPOUNDS BY MOHAMAD HADZRI YAACOB PS 023001

INTRODUCTION Mycotoxin group Produced by two fungi; Aspergillus flavus

Aspergillus flavus and Aspergillus parasiticus Types of aflatoxins: AFB1, AFB2, AFG1, AFG2 AFM1 and AFM2 3/1/2007

PhD viva presentation

Contaminated corn

INTRODUCTION…cont

Chemical structures; O

O

O

AFB1

O

AFB2 O

O

O

O

O

O

O

O O

O

O

AFG1 O

O

O

O

O O

3/1/2007

AFG2

O

O

O

O

PhD viva presentation

O

OBJECTIVE • To study electroanalytical properties of aflatoxin compounds (AFB1, AFB2, AFG1 and AFG2) at the working electrode. • To develop a new alternative technique for high sensitive determination of aflatoxin compounds

3/1/2007

PhD viva presentation

SCOPE OF STUDY • Studies on the voltammetric behaviour of aflatoxins using cyclic voltammetric (CV) technique. • Studies on the differential pulse cathodic and anodic stripping voltammetry of all aflatoxins and parameters optimisation • Studies on the effect of increasing concentration of the aflatoxins to their peak height 3/1/2007

PhD viva presentation

SCOPE OF STUDY…cont • Interference studies by metal ions and organic compounds • Studies on the square wave stripping voltammetry technique for the determination of aflatoxin compounds • Application of the proposed methods for the determination of aflatoxin compounds in real samples. The results were compared with HPLC results • Stability studies of aflatoxin solutions 3/1/2007

PhD viva presentation

RESULTS AND DISCUSSION • By CV technique; 25 20 I p (n A )

¾ Britton Robinson Buffer (BRB) at pH 9.0 was the best supporting electrolyte

30

15 10 5 0

For 0.6 µM AFB1; pH = 9.0, Ip = 24.5 nA 3/1/2007

PhD viva presentation

5

6

7

8

9

10

pH of BRB

11

12

13

RESULTS AND DISCUSSION…cont • Suggested mechanism (Smyth et al.,1979) O

O

O

O

O

O

2e -

2

2

OCH 3

OCH 3

2 H 2O

Dimer

3/1/2007

PhD viva presentation

+ 2 OH -

RESULTS AND DISCUSSION…cont ¾ All aflatoxin compounds are undergo irreversible reduction reaction at mercury electrode

For 1.3 µM AFB2; Ip = 30 nA Ep = -1.315 V (Ag/AgCl) 3/1/2007

PhD viva presentation

RESULTS AND DISCUSSION…cont ¾ All aflatoxin compounds adsorb on the surface of the mercury electrode. 60 50 I p (n A )

40 30 20 10 0 1

2

3 No of cycle

Repetitive CV; Ip increased with the number of cycle 3/1/2007

PhD viva presentation

4

5

RESULTS AND DISCUSSION…cont • From DPCSV experiment ¾ Optimised parameters: Ei = -1.0 V (except for AFG1; -0.95 V), Ef = -1.4 V, Eacc = -0.6 V, tacc = 80 s, scan rate = 50 mV/s and pulse amplitude = 80 mV. 0.06 0.06µM µMAFB2 AFB2

0.06 µM AFB2 in BRB at pH 9.0 (a) Unoptimised (b)Optimised

3/1/2007

PhD viva presentation

RESULTS AND DISCUSSION…cont ¾

Method validation

LOD/ppb

AFB1 1.56

AFB2 2.50

LOQ/ppb

5.20

8.33

Linearity Range/ ppb

6 – 100

6 – 100 6 – 105

6 – 105

R2

0.9989

0.9980

0.9989

3/1/2007

PhD viva presentation

AFG1 3.28 10.93

0.9992

AFG2 2.50 8.33

RESULTS AND DISCUSSION…cont ¾Method validation Accuracy: % Recovery > 95% Î High accuracy Precision: %RSD for n = 8 < 3.0% Î High precsion 3/1/2007

PhD viva presentation

RESULTS AND DISCUSSION…cont Robustness; Small influences of important conditions (pH of BRB, Eacc and tacc ) were not significantly affect the peak height Î Method is robust Ruggedess; No significant different in the peak heights obtained by using two different voltammetric analysers (BAS vs Metrohm) Î Method is rugged 3/1/2007

PhD viva presentation

RESULTS AND DISCUSSION…cont • Interference studies; Metal ions (Al, Cu, Pb, Ni and Zn) and organic compounds (ascorbic acid, β-cyclodextrin and Lcysteine) were not interfere up to 10 times concentration as compared with aflatoxin compounds

3/1/2007

PhD viva presentation

RESULTS AND DISCUSSION…cont Using SWSV technique • For all aflatoxins, the peak heights were increased > 10 times Î More sensitive • Analysis can be completed within a few second Î Faster technique • LOD and LOQ lower than DPCSV technique Î More sensitive

3/1/2007

PhD viva presentation

RESULTS AND DISCUSSION…cont • Voltammograms of aflatoxins (DPCSV vs SWSV)

AFB1 3/1/2007 page no - 191 Text:

AFB2

AFG1 PhD viva presentation

AFG2

RESULTS AND DISCUSSION…cont • From stability studies; 10 ppm aflatoxin stock solutions (in benzene:acetonitrile, 98%) stored in the dark and cool condition: stable up to 1 year. 1 ppm aflatoxin standard solutions (in BRB pH 9.0 ) stored in the dark and cool conditions: stable up to 3 months for AFB1 and AFB2 while for AFG1 and AFG2 stable up to 2 months.. 3/1/2007

PhD viva presentation

RESULTS AND DISCUSSION…cont 1 ppm aflatoxins in BRB pH 9.0 exposed to normal laboratory condition; AFB1 and AFB2 were stable up to 8 hrs but for AFG1 and AFG2 were stable up to 3 hrs. Aflatoxin prepared in basic condition were less stable as compared to those prepared in acidic and neutral media.

3/1/2007

PhD viva presentation

RESULTS AND DISCUSSION…cont • Analysis real sample (groundnuts) Followed extraction and clean-up procedure as applied by Chem. Dept. MOSTI. Recovery studies: Aflatoxins standard solutions were spiked into the groundnut eluates; 80-99% for 1st day analysis, 80 -90% for 2nd day and 7585% for 3rd day analysis. 3/1/2007

PhD viva presentation

RESULTS AND DISCUSSION…cont • Analysis of 15 samples by DPCSV, SWSV and HPLC (result: total aflatoxins in ppb level) Sample

DPCSV

SWSV

HPLC

Notes

S01

12.73

13.02

14.34

-ve

S02

7.12

8.36

8.83

-ve

S03

n.d

n.d

0.50

-ve

S04

21.46

29.72

19.08

+ve

3/1/2007

PhD viva presentation

RESULTS AND DISCUSSION…cont Sample

DPCSV

SWSV

HPLC

S05

9.90

8.95

5.34

-ve

S06

n.d

n.d

n.d

-ve

S07

5.90

8.17

3.67

-ve

S08

n.d

n.d

n.d

-ve

3/1/2007

PhD viva presentation

Notes

RESULTS AND DISCUSSION…cont Sample

DPCSV

SWSV

HPLC

S09

n.d

n.d

1.84

-ve

S10

31.93

35.63

36.00

+ve

S11

n.d

n.d

0.42

-ve

S12

n.d

n.d

1.75

-ve

3/1/2007

PhD viva presentation

Notes

RESULTS AND DISCUSSION…cont Sample

DPCSV

SWSV

HPLC

S13

10.76

9.21

8.25

-ve

S14

n.d

n.d

n.d

-ve

S15

n.d

n.d

n.d

-ve

+ve : > 15 ppb ( 2 out of 15 samples: 13%) 3/1/2007

PhD viva presentation

Notes

CONCLUSIONS The study has achieved the objective; A sensitive, accurate, robust, rugged, fast and low cost technique was successfully developed and applied for the determination of aflatoxins in groundnut samples The hypothesis regarding electroanalytical properties of aflatoxins was also proven. 3/1/2007

PhD viva presentation

SUGGESTIONS • Use other types of the working electrode (CPE, GCE or BFE) • Study the reagents that can be complexed with aflatoxins • Study the effect of storage time and conditions of groundnuts to the level of aflatoxins • Analyse aflatoxins in other type of samples (rice, bread, fish, vegetables and cigarretes tobacco) using the proposed technique. 3/1/2007

PhD viva presentation

ACKNOWLEDGEMENT • Supervisors: AP Dr Abdull Rahim Hj. Mohd. Yusoff and Prof Rahmalan Ahamad • USM: study leave and fellowship • UTM: Short term grant (Vot No: 75152/2004) • Chemistry Dept staff 3/1/2007

PhD viva presentation

3/1/2007

PhD viva presentation

ORAL PRESENTED AT KUSTEM 3rd ANNUAL SEMINAR ON SUSTAINABLE SCIENCE AND MANAGEMENT (TERENGGANU; 4 - 5th MAY 2004)

CYCLIC VOLTAMMETRIC STUDY OF AFLATOXIN G1 (AFG1) AT THE MERCURY ELECTRODE BY Mohd Hadzri Yaacob, Abd.Rahim Yusoff and Rahmalan Ahamad UTM, SKUDAI

TOPICS FOR DISCUSSION • INTRODUCTION • OBJECTIVE • EXPERIMENTAL • RESULTS AND DISCUSSION • CONCLUSION • ACKNOWLEDGEMENT

AFLATOXINS

INTRODUCTION • AFLATOXINS: Mycotoxin group. Produced by two fungi; Aspergillus flavus

Types of aflatoxins: AFB1, AFB2, AFG1 and AFG2:

Aspergillus parasiticus

Raw peanuts, peanuts products, wheat, maize, corn, vegetable oils, dried fruites, barley, pear, cocoa, coffee, herbs, spices, wine, cottonseeds.

Cow milk and its products, eggs

AFM1 and AFM2 and meat product

INTRODUCTION …cont • AFLATOXINS: Occurs naturally in commodities used for animal and human Carcinogenic, teratogenic and mutagenic IARC (1988): Class I carcinogenic materials

Dangerous to health: causes aflatoxicosis disease. Effect the economy of commodities export countries

AFLATOXIN G1 ( AFG1): Chemical and physical properties O

O

O

C17H12O7 O

MW = 328

O O

O

Chemical name: 3,4,7a, 10a-tetrahydro-5-methoxy-1H, 12H furo[3’,2’:4,5]furo[2,3-h]pyrano[3,4-c][l]-benzopyran1,12-dione Yellowish crystal. Odorless, tasteless and colorless solution ( in organic solvent).

AFLATOXIN G1 ( AFG1): Chemical and physical properties…cont Solubility: Dissolve in methanol, water:acetonitrile (9:1), trifluoroactic acid, DMSO and acetone. In water: 10-20 mg / liter m.p; 244 – 246 0 C Produces green color under UV light UV abs (max) in methanol: 216, 242, 265 and 362 nm IR spectrum ( in chloroform): 1760, 1695, 1630 and 1595 cm-1

Stability of AFG1 In crystal form: extremely stable in the absence of light or UV radiation, even at T = > 1000C Solution in water, DMSO, 95% acetone or ethanol stable for 24 hrs under normal condition. Stable for years if kept in cool and dark place Stable in most food process; not destroy by normal cooking process since it heat stable

ELECTROCHEMICAL PROPERTIES OF AFG1 Based on chemical structure: Ketone groups in lactone ring can be reduced and produce cathodic wave. Unsaturated terminal furan ring can be reduced and produce cathodic wave. O

O

O

O

O O

O

Can forms anionic radical

WHAT AND WHY CYLIC VOLTAMMETRY (CV)? CV: Voltammetric technique that consist of scanning linearly the potential of a stationary working electrode ( forward and reverse ) and the current flowing through the cell is measured as a function of that potential. Three parameters needed to be characteristiced; starting potential of the scan, finishing / switching potential and scan rate ( rate of change of potential with the time)

A plot of current as a function of applied potential is called a voltammogram ( as in figure below)

Typical cyclic voltammogram for a reversible redox process O = oxidised form

R = reduced form

Why CV? 1. To aquire qualitative information about electrochemical reactions 2. Ability to rapidly provide information on; @ thermodynamic of redox process @ the kinetic of heterogeneous electron-transfer @ coupled chemical reactions @ adsorption processes at electrode surface 3. Offer a rapid location of redox potentials of the electroactive species and convenient for evaluation of the effect of media upon the redox process Î Initial experiment performed in electroanalytical study

OBJECTIVE

1) To get information about electrochemical properties of aflatoxin G1 (AFG1) on the mercury electrode. 2) From this information, other voltammetric technique can be developed ( such as differential pulse stripping voltammetric ) for quantitatively determination of AFG1 ( AFG1 standard or AFG1 in real samples)

EXPERIMENTAL: INSTRUMENTS AND REAGENTS

BAS C50W Voltammetric Analyser

BAS CGME consist of Ag/AgCl as RE, Pt wire as AE, CGME as WE, 20 ml cell and magnetic stirrer.

Reagents: AFG1 ( MERCK); 10 ppm in benzene: acetonitrile (98%), 1 ppm: prepared in BRb pH 9.0.

EXPERIMENT A) Cathodic and anodic scan of AFG1

1.

10 ml of supporting electrolyte (BRb pH = 9.0 ) in voltammetric cell was degassed for at least 10 min.

2.

Scanned using CV technique; Ei = 0 mV, E high = 0 mV, E low = 1500 mV, scan rate = 200 mV/s to obtain voltammogram of blank.

3.

The AFG1 standard solution was spiked into the cell ( concentration of AFG1 in the cell = 1.5 uM), was degassed for at least 3 min and scanned follows the same procedure as 2.0 to obtain voltammogram of AFG1 in BRb solution.

4.

Experiment no 2 and 3 were repeated with different parameters ( Ei = -1500 mV, E high = 0 mV and E low = -1500 mV ). Other parameters remain unchanged.

EXPERIMENT…Cont B) Repeatitive scan Repeatitive scan ( n = 5 ) for cathodic direction was performed and the peak height of AFG1 was observed C) Standard Addition of AFG1 Using the same parameters as no 2, the concentration of AFG1 was added ( 1.5, 2.25 , 3.0, 3.75 and 4.5 M). The peak current and peak potential of AFG1 was observed. D) Effect of scan rate The scan rate was changed ( 25, 50, 100, 150, 200, 250, 300, 400, 500, 700 mV/s ) while other parameters remain unaltered. The peak current and peak potential of AFG1 was observed due to the various applied scan rate.

EXPERIMENT…Cont

E) Effect of pH of BRb solution The pH of supporting electrolyte (BRb solution ) was adjusted from 3.0 to 13.0 ( using 1.0 M NaOH or 1.0 M HCl ) and the AFG1 standard solution was scanned in various pH of BRB. The peak current and peak potential of AFG1 was observed.

RESULTS AND DISCUSSION A) Cathodic and anodic scan of AFG1 Ip = 45.2 nA, Ep = -1245 mV Ei = 0 mV, E high = 0 mV, E low = -1500 mV, s.rate = 200 mV/s, [AFG1] = 1.5 uM in BRb pH =9.0 Ip = 58.5 nA, Ep = -1243 mV Ei = -1500 mV, E high = 0 mV, E low = -1500 mV, s.rate = 200 mV/s, [AFG1] = 1.5 uM Both scan directions gave only single cathodic peak ( no anodic wave ) ÎAFG1 undergoes irreversible reduction reaction at mercury electrode.

RESULTS AND DISCUSSION… cont B) Repeatitive cathodic scan ( n = 5 or 10 segments) 80

Ip (n A )

60 40 20 0 1

2

3 No of cycle

Peak heights of AFG1 increased with the number of scans with no extra peak neither anodic nor anodic peak was observed. Peak potentials of AFG1 were – 1245 to -1220 mV. =Î Progressive adsorptive accumulation of AFG1 at the mercury electrode surface

4

5

RESULTS AND DISCUSSION… cont C) Standard Addition of AFG1 150

Ip (nA)

120 90 60 y = 24.756x + 13.072

30

R2 = 0.9812

0 1

1.5

2

2.5

3

3.5

4

4.5

5

[AFG1] / uM

Ei = 0 mV, E high = 0 mV, E low = -1500 mV, s.rate = 200 mV/s, [AFG1] = 1.5, 2.25, 3.0, 3.75 and 4.5 uM in BRB pH =9.0

Ip of AFG1 increased and peak potentials shifted toward less negative direction with increasing of AFG1 concentration.

The peak potential at –1245 mV was confirmed due to the reduction of AFG1 in BRB pH =9.0.

RESULTS AND DISCUSSION… cont D) Effect of scan rate

Lop Ip

i) Log peak height versus log scan rate, [AFG1] = 1.5 uM 2.1 1.9 1.7 1.5 1.3 1.1 0.9 0.7 0.5

y = 0.5623x + 0.446 R2 = 0.9919

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

log sca n ra te

Linear relationship was observed between log Ip versus log scan rate with the slope of 0.56 ( > 0.5 ) =Î Diffusion current is influenced by an adsorption of AFG1 on the electrochemical process at mercury electrode surface.

RESULTS AND DISCUSSION… cont ii) Peak potential versus log scan rate, [AFG1] = 1.5 uM 1275

Ep (-mV)

1260 1245 1230 y = 48.484x + 1134.8 R2 = 0.9978

1215 1200 1185 1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

log scan rate

Plot of peak potential versus log scan rate is a linear graph ( R2 = 0.9978) =Î confirmed that the reduction of AFG1 on the electrode surface is totally irreversibe.

RESULTS AND DISCUSSION… cont

Ip (nA)

iii) Peak height versus scan rate, [AFG1] = 1.5 uM 110 100 90 80 70 60 50 40 30 20 10 0

1) Slow scan rate: Ip = 0.2298x + 14.164 ( R2 = 0.9757, n = 5)

2) Fast scan rate: 0

50 100 150 200 250 300 350 400 450 500 550

Ip = 0.1598x + 19.18 ( R2 = 0.9892, n = 5)

Scan rate ( m V/s )

Based on the slopes of the regression equations for two different regions, the reduction and adsorption of AFG1 on the electrode surface is governed by the speed of reaction which preferred slow reaction.

RESULTS AND DISCUSSION… cont E) Effect of different pH of BRb solution, [AFG1] = 1.5 uM

Ip (nA )

i) Effect on peak height 60 50 40 30 20 10 0

At pH = 9.0, highest peak current was observed

2

3

4

5

6

7

8

9

10 11 12

ii) Effect on peak potential

pH 1270

Peak height and peak potential of AFG1 are pH dependent except for pH 6,7, 8 and 9, peak potential are unchanged at -1245 mV

Ep (-mV)

1250 1230 1210 1190 1170 1150 2

3

4

5

6

7 pH

8

9

10

11

12

CONCLUSION 1. AFG1 is reduced at mercury electrode. The reduction reaction is totally irreversible reaction which gives maximum peak height in BRb at pH of 9.0. The diffusion current produced by this reaction is affected by adsorption process of AFG1 at the mercury electrode surface which prefer a slow reaction. 2. All information on electrochemical properties of AFG1 are useful for development of other voltammetric technique for determination of AFG1 in standard solution and real samples.

ACKNOWLEDGEMENT SUPERVISOR: AP DR ABDUL RAHIM MOHD YUSOFF CO-SUPERVISORS: 1. PROF RAHMALAN AHAMAD 2. AP DR ABD. RAHIM YAACOB ELECTROANALYSIS GROUP AND STAFF OF CHEMISTRY DEPT

SCHOOL OF HEALTH SCIENCES, USM , HEALTH CAMPUS, KUBANG KRIAN, KELANTAN

THANK YOU

Proposed mechanisme of the reduction of AFG1 that may take place at mercury electrode as reported by Smyth et al. (1979);

O

O

O

2

O O

O

2e-

ΘO

O

O

O O

O

O

O

2H2O

Dimer + 2OH-

ORAL PRESENTED AT SYMPOSIUM LIFE SCIENCE II (USM PENANG; 31st MAC – 3rd APRIL 2004)

3/1/2007

2nd Life PostCon USM Penang

1

STABILITY STUDY OF AFLATOXIN G1 (AFG1) USING DIFFERENTIAL PULSE STRIPPING VOLTAMMETRIC TECHNIQUE BY Mohd Hadzri Yaacob, Abd.Rahim Yusoff and Rahmalan Ahamad UTM, SKUDAI

3/1/2007

2nd Life PostCon USM Penang

2

TOPICS FOR DISCUSSION • INTRODUCTION

AFLATOXINS

• OBJECTIVE • EXPERIMENTAL • RESULTS AND DISCUSSION • CONCLUSION • ACKNOWLEDGEMENT 3/1/2007

2nd Life PostCon USM Penang

3

INTRODUCTION • AFLATOXINS: Mycotoxin group. Produced by two fungi; Aspergillus flavus

Types of aflatoxins: AFB1, AFB2, AFG1 and AFG2:

Aspergillus parasiticus

Raw peanuts, peanuts products, wheat, maize, corn, vegetable oils, dried fruites, barley, pear, cocoa, coffee, herbs, spices, wine, cottonseeds.

Cow milk and its products, eggs

AFM1 and AFM2 and meat product

3/1/2007

2nd Life PostCon USM Penang

4

INTRODUCTION …cont • AFLATOXINS: Occurs naturally in commodities used for animal and human Carcinogenic, teratogenic and mutagenic IARC (1988): Class I carcinogenic materials

Dangerous to health: causes aflatoxicosis disease. Effect the economy of commodities export countries 3/1/2007

2nd Life PostCon USM Penang

5

AFLATOXIN G1 ( AFG1): Chemical and physical properties O

C17H12O7

O

O

O

MW = 328

O O

O

Chemical name: 3,4,7a, 10a-tetrahydro-5-methoxy-1H, 12H furo[3’,2’:4,5]furo[2,3-h]pyrano[3,4-c][l]-benzopyran1,12-dione Yellowish crystal. Odorless, tasteless and colorless solution ( in organic solvent). 3/1/2007

2nd Life PostCon USM Penang

6

AFLATOXIN G1 ( AFG1): Chemical and physical properties…cont Solubility: Dissolve in methanol, water:acetonitrile (9:1), trifluoroactic acid, DMSO and acetone. In water: 10-20 mg / liter m.p; 244 – 246 0 C Produces green color under UV light UV abs (max) in methanol: 216, 242, 265 and 362 nm IR spectrum ( in chloroform): 1760, 1695, 1630 and 1595 cm-1 3/1/2007

2nd Life PostCon USM Penang

7

Stability of AFG1 In crystal form: extremely stable in the absence of light or UV radiation, even at T = > 1000C Solution in water, DMSO, 95% acetone or ethanol stable for 24 hrs under normal condition. Stable for years if kept in cool and dark place Stable in most food process; not destroy by normal cooking process since it heat stable

3/1/2007

2nd Life PostCon USM Penang

8

Stability of AFG1… cont Based on chemical structure: In strong alkali: lactone ring- susceptible to alkaline hydrolysis In acid: acid catalytic addition of water across the double bond of the furan ring. O

O

O

O

O O

3/1/2007

O

2nd Life PostCon USM Penang

9

OBJECTIVE To study the stability of AFG1 in; a) Britton-Robinson buffer (BRB) pH 9.0; AFG1 prepared in BRB pH 9.0, measured in same buffer, exposed to normal condition ( 0 to 6 hrs). b) BRB pH 9.0: kept in cool and dark place ( 0 to 6 months ) c) AFG1 prepared in BRB pH 9.0, measured in BRB with different pH, expose to normal condition ( 0 to 3 hrs). 3/1/2007

Using Differential pulse cathodic stripping 2ndtechnique Life PostCon USM (DPCSV) Penang voltammetric

10

VOLTAMMETRY: From Voltage- Amperometry -Measure amount of current at different voltage -Electroanalytical technique : sensitive, high accuracy, low cost and easy to use -2 important parameters; peak height (Ip) and peak potential (Ep) -result: voltammogram ( e.g; Differential Pulse Cathodic Stripping Voltammogram of AFG1)

[AFG1] = 0.1 uM = 30 ppb in BRB pH 9.0 Ep = -1150 mV Ip = 55.45 nA Analysis date: 26th Jan 2004 3/1/2007

2nd Life PostCon USM Penang

11

EXPERIMENTAL: INSTRUMENTS AND REAGENTS

BAS C50W Voltammetric Analyser

BAS CGME consist of Ag/AgCl as RE, Pt wire as AE, CGME as WE, 20 ml cell and magnetic stirrer.

Reagents: AFG1 ( MERCK); 10 ppm in benzene: acetonitrile (98%), 1 ppm: prepared in BRB pH 9.0. BRB pH ( 3.0 to 13.0) 3/1/2007 2nd Life PostCon USM Penang 12

EXPERIMENTAL FLOW CHART

10 ML BRB SOLUTION

NITROGEN GAS IS FLOWED IN AFLATOXIN G1 SOLUTION, REDISSOLVED IN BRB SOLUTION

SPIKE INTO 20 ML VOLTAMMETRIC CELL

SOLUTION IS DEGASSED

BLANK SOLUTION BLANK VOLTAMMOGRAM 3/1/2007

SOLUTION IS SCANNED OBTAINED VOLTAMMOGRAM 2nd Life PostCon USM Penang

BLANK SOLUTION + AFLATOXIN SAMPEL VOLTAMMOGRAM 13

RESULTS a) 0.1 uM AFG1 in BRB pH =9.0, exposed to normal condition Stability study of AFG1 in BRB pH 9

Ip (n A )

80 60 40 20 0 0

1

2

3

4

5

6

7

8

Standing time (hrs)

0 to 3 hrs: Peak current: 60.26 to 57.7 nA 4 hrs: 43.67 nA ( 78%) 6 hrs: 33.56 nA ( 56%) 3/1/2007 8 hrs:

AFG1 stable up to 3 hrs in BRB pH 9.0, exposed to normal condition

2nd Life PostCon USM Penang 29.02 nA ( 45%)

14

b) 0.1 uM AFG1 from 1 ppm AFG1 in BRB pH 9.0 kept in cool and dark place, measured in BRB pH 9.0 60

Ip (nA)

50 40 30 20 10 0 0

1

2

3

4

5

6

Storage time (month)

(a) 0 (b) 1 m (c) 2 m (d) 3 m (e) 4 m (f) 5 m (g) 6 m

0 to 3 m : 57.50 – 39.50 nA ( 70%) 4 m : 36.5 nA (60%) 3/1/2007

6 m ( 25%) 2nd Life PostCon USM Penang

AFG1 in BRB pH 9.0, kept in cool and dark place Îstable up to 3 m keeping time 15

c) 0.1 uM AFG1 ( from 1 ppm AFG1 in BRB pH 9.0) measured in different pH of BRB and exposed to normal condition 70 60

Ip (nA)

50

pH = 6.0

40

pH = 7.0

30

pH = 9.0 pH = 11.0

20 10

Stability order for AFG1 in different pH of BRB exposed to normal condition up to 3 hrs;

0 0

1

2

Standing tim e (hr s )

3

pH 11.0 < 9.0 < 6.0 < 7.0

In BRB pH = 6.0, 7.0 : peak current increases with time In BRB pH = 9.0: peak current slowly decreases with time In BRB pH = 11.0 peak current sharply decreases with 3/1/2007where after 2 hrs 2nd Life Penang time noPostCon peakUSM was observed

16

CONCLUSION 1) AFG1 stable up to 3 hrs in BRB pH 9.0 when exposed to normal condition. 2) AFG1 in BRB pH 9.0, stable up to 3 m when kept in cool and dark place 3)Stability order for AFG1 in different pH of BRB exposed to normal condition up to 3 hrs; pH 11.0 < 9.0 < 6.0 < 7.0

3/1/2007

2nd Life PostCon USM Penang

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FURTHER WORKS 1. Stability study of AFG1 using different concentration of AFG1 ( 0.05 uM and 1.0 uM) 2. Stability study of AFG1, keep in room temperature and expose to light from 0 to 6 months. 3. Stability study of other aflatoxins (AFB1, AFB2 and AFG2)

3/1/2007

2nd Life PostCon USM Penang

18

ACKNOWLEDGEMENT SUPERVISOR: AP DR ABDUL RAHIM MOHD YUSOFF CO-SUPERVISORS: 1. PROF RAHMALAN AHAMAD 2. AP DR ABD. RAHIM YAACOB ELECTROANALYSIS GROUP AND STAFF OF CHEMISTRY DEPT

SCHOOL OF HEALTH SCIENCES, USM ( STUDY LEAVE AND FELLOWSHIP UNDER ASHES: May 2002 – May 2005) MR RODWAN MAT AIL, CHEM DEPT, MOSTE, PENANG.

3/1/2007

2nd Life PostCon USM Penang

19

THANK YOU Wassalam Mohd Hadzri, Hadzri, Sains Forensik, Forensik, PPSains Kesihatan USM KELANTAN

3/1/2007

2nd Life PostCon USM Penang

20

ORAL PRESENTED AT SKAM-17 (KUANTAN, 24-26th AUGUST 2004)

STABILITY STUDIES OF AFLATOXINS USING DIFFERENTIAL PULSE CATHODIC STRIPPING VOLTAMMETRIC (DPCSV) TECHNIQUE BY Mohd Hadzri Yaacob, Abd.Rahim Yusoff and Rahmalan Ahamad UTM, SKUDAI

INTRODUCTION AFLATOXINS

C=O groups

Electroactive

Electroactive property of aflatoxins

species can be determined using voltammetric technique

INTRODUCTION…cont • AFLATOXINS: Mycotoxin group. Produced by two fungi; Aspergillus flavus

Types of aflatoxins: AFB1, AFB2, AFG1 and AFG2:

Aspergillus parasiticus

Raw peanuts, peanuts products, wheat, maize, corn, vegetable oils, dried fruites, barley, pear, cocoa, coffee, herbs, spices, wine, cottonseeds.

Cow milk and its products, eggs

AFM1 and AFM2 and meat product

INTRODUCTION …cont • AFLATOXINS: Occurs naturally in commodities used for animal and human Carcinogenic, teratogenic and mutagenic IARC (1988): Class I carcinogenic materials

Dangerous to health: causes aflatoxicosis disease. Effect the economy of commodities export countries

AFLATOXIN: Chemical and physical properties O

O

AFB1

O

O

AFB2 O

O

O

O

O

O

O

O

C17H12O6,

MW = 312 O

C17H14O6

MW = 314

O

O

AFG1

O

AFG2 O

O

O

O

O

O O

O

C17H12O7 MW = 328

O

C17H14O7

O

MW = 330

AFLATOXIN: Chemical and physical properties…cont

Solubility: Dissolve in methanol, water:acetonitrile (9:1), trifluoroactic acid, DMSO and acetone. In water: 10-20 mg / liter Yellowish crystal Odorless, tasteless and colorless solution ( in organic solvent).

AFLATOXIN: Chemical and physical properties…cont

m.point

AFB1

AFB2

AFG1

AFG2

2690 C

2870C

2450C

2370C

UV abs (max) 360 in methanol/

362

362

362

nm

IR spectrum 1760, 1684 ( in chloroform) 1632, 1598 / cm-1 1562

1760, 1684 1632, 1598 1562

1760, 1695 1630, 1595

1760, 1695 1630, 1595

Stability of aflatoxins In crystal form: extremely stable in the absence of light or UV radiation, even at T = > 1000C Solution in water, DMSO, 95% acetone or ethanol: stable for 24 hrs under normal condition. Stable for years if kept in cool and dark place

Stable in most food process; not destroy by normal cooking process since it heat stable.

OBJECTIVE To study the stability of aflatoxins which were; a) exposed to normal laboratory condition for 8 hours in BRb pH=9.0. b) exposed to normal laboratory condition for 3 hours in BRb pH=6.0, 7.0, 9.0 and 11.0 c) stored under cool temperature for 6 months in BRb pH =9.0 using DPCSV technique ( a small portion of my research study in developing voltammetric technique for determination of aflatoxins)

EXPERIMENTAL: INSTRUMENTS AND REAGENTS

BAS C50W Voltammetric Analyser

BAS CGME consist of Ag/AgCl as RE, Pt wire as AE, CGME as WE, 20 ml cell and magnetic stirrer.

Reagents: AFB1, B2, G1, G2 ( MERCK); 10 ppm in benzene: acetonitrile (98%), 1 ppm: prepared in BRb at required pH.

EXPERIMENT A) General procedure for DPCSV analysis 1. 10 ml of supporting electrolyte (BRb pH = 9.0 ) in voltammetric cell was degassed for at least 15 min. 2. Scanned using DPCSV technique; Ei = -1000 mV, Ef = 1400 mV, Eacc = -600 mV, tacc = 80 s, scan rate = 50 mV/s, pulse amplitude = 80 mV and rest time = 10 s to obtain voltammogram of blank ( for all aflatoxins except for AFG1, Ei = -950 mV) 3. The aflatoxins standard solution was spiked into the cell ( concentration of aflatoxins in the cell = 0.1 uM), redegassed for at least 2 min and rescanned follows the same procedure as no 2.0 to obtain voltammograms of aflatoxins in BRb solution.

EXPERIMENT…Cont B) Procedures for stability studies 1) 8 hours exposed to normal condition Each aflatoxin was scanned every hour from 0 to 8 hours. ( aflatoxin in BRb pH =9.0 solution remained in the voltammetric cell). 2) 3 hours exposed to normal condition Each aflatoxin was scanned every hour from 0 to 3 hours in different pH of BRb ( 6.0, 7.0, 9.0 and 11.0) 3) Shelf life of aflatoxins ( 1 ppm) stored in freezer Each aflatoxin ( 0.1 uM) was scanned every month from 1st to 6th month storage period in BRb pH =9.0

RESULTS AND DISCUSSION A) Voltammograms of aflatoxins AFB1

[AFB1] = 0.1 uM / 31.2 ppb In BRb pH =9.0 blank

Ep = -1210 mV Ip = 63.19 +/- 1.64 nA n=8

RSD = 2.60%

[AFB2] = 0.1 uM / 31.5 ppb In Brb pH 9.0

AFB2

blank

Ep = -1230 mV Ip = 65.67 +/- 1.28 nA n=8

RSD = 1.95%

RESULTS AND DISCUSSION…cont AFG1

[AFG1] = 0.1 uM / 32.8 ppb In BRb pH 9.0 Ep = -1150 mV

blank

Ip = 60.76 +/- 0.99 nA n=8

RSD = 1.63%

[AFG2] = 0.1 uM / 33.0 ppb In BRb pH 9.0

AFG2

Ep = -1170 mV blank

Ip = 58.88 +/- 0.88 nA n=8

RSD = 1.50%

RESULTS AND DISCUSSION… cont ( STABILITY STUDIES) A) 8 hours exposed to normal laboratory condition 70 Peak height (nA)

60 50

AFB1

40

AFB2

30

AFG1

20

AFG2

10 0 0

1

2

3

4

5

6

7

8

9

Exposure time (hrs)

Peak currents of AFB1 and AFB2 remained unchanged up to 8 hours but for AFG1 and AFG2 peak currents started decrease after 1st hour exposure time. After 3 hours it gradually decreased with AFG2 gave lower peak current compared to AFG1. ÎUp to 8 hrs, AFB1=AFB2 > AFG1, AFG2. AFG1> AFG2

RESULTS AND DISCUSSION… cont ( STABILITY STUDIES) Voltammograms of aflatoxins at 0 to 8 hours in BRb pH 9.0.

0.1 uM AFB1

0.1 uM AFB2

RESULTS AND DISCUSSION… cont ( STABILITY STUDIES) Voltammograms of aflatoxins at 0 to 8 hours in BRb pH 9.0...cont

AFG1 = 0.1 uM

AFG2 = 0.1 uM

RESULTS AND DISCUSSION… cont (STABILITY STUDIES)

B) In different pH of BRb ( pH 6.0 and (7.0) 50

70

pH = 6.0

45

pH = 7.0

60

35 AFB1

30

AFB2

25

AFG1

20

AFG2

15 10

P eak h eig h t (n A)

P eak h eig h t (n A)

40 50 AFB1 40

AFB2

30

AFG1 AFG2

20 10

5

0

0 0

1

2

3

Exposure time (hrs)

In BRb pH = 6.0

0

1

2

3

Exposure time (hrs)

In BRb pH = 7.0

In pH 6.0 and 7.0, all aflatoxins are stable within 3 hours in the cell.

RESULTS AND DISCUSSION… cont (STABILITY STUDIES)

B) In different pH of BRb ( pH 9.0 and 11.0) Stability of aflatoxins in BRB pH 9

Stability of aflatoxins in BRB pH 11

70

45

60

40 35

50 AFB2

30

AFG1 AFG2

20

Ip (nA)

Ip (nA)

AFB1 40

30

AFB1

25

AFB2

20

AFG1

15

AFG2

10

10

5

0

0

0

1

2

3

Exposure time (hrs)

In BRb pH 9.0 In pH 9.0, all aflatoxins stable within 3 hours in the cell.

0

1

2

3

Exposure time (hrs)

In BRb pH11.0 In pH 11.0, AFG1 and AFG2 significantly decreased within 3 hours compared to AFB1 and AFB2.

RESULTS AND DISCUSSION… cont (STABILITY STUDIES) B) 6 months kept in freezer AFG1

AFB2 80

Peak height (nA)

70 60 AFB1 50

AFB2

40

AFG1 AFG2

30 20 10 0

1

2

3

4

5

6

7

Storage time in freezer (months)

AFB1

AFG2 Stability order: AFB1=AFB2>AFG2>AFG1 ( in BRb pH =9.0)

RESULTS AND DISCUSSION… cont (STABILITY STUDIES)

80

100

70

90

60

80 AFB1

50

AFB2

40

AFG1 AFG2

Percent left

Peak height (nA)

B) 6 months kept in freezer

AFB1 70

AFB2

60

AFG1 AFG2

30

50

20

40

0.1 uM in BRb pH = 9.0

10

30 0

1

2

3

4

5

6

7

Storage time in freezer (months)

Peak height vs time

0

1

2

3

4

5

6

7

Storage time in freezer (months)

Percent left vs time

3rd MONTH: AFB1 (72%), AFB2 (83%), AFG1 (66%), AFG2 (56%) 6th MONTH: AFB1 (50%), AFB2 (59%), AFG1 (42%), AFG2 (37%)

CONCLUSION 1. In BRb pH 9.0, AFB1 and AFB2 are stable up to 8 hours while AFG1 and AFG2 are stable up to 3 hours when exposed to normal condition in voltammetric cell. 2. Within 3 hours exposed to normal condition, all aflatoxins are stable in BRb pH 6, 7 and 9.0 but in BRb pH 11.0, AFG1 is very unstable compared to other aflatoxins. The proposed stability order for aflatoxins in BRb pH 11.0 is: AFB2 > AFB1 > AFG2 > AFG1

CONCLUSION…. cont

3. AFB1 and AFB2 solution in BRb pH 9.0 kept in freezer have shelf life of 3 months while AFG1 and AFG2 only 2 months. After this period of time, the new aflatoxins solution should be prepared.

ACKNOWLEDGEMENT SUPERVISOR: AP DR ABDUL RAHIM MOHD YUSOFF CO-SUPERVISORS: 1. PROF RAHMALAN AHAMAD 2. AP DR ABD. RAHIM YAACOB ELECTROANALYSIS GROUP AND STAFF OF CHEMISTRY DEPT

SCHOOL OF HEALTH SCIENCES, USM , HEALTH CAMPUS, KUBANG KRIAN, KELANTAN

THANK YOU

Stability of AFG1… cont Based on chemical structure: In strong alkali: lactone ring- susceptible to alkaline hydrolysis In acid: acid catalytic addition of water across the double bond of the furan ring. O

O

O

O

O O

O

ELECTROCHEMICAL PROPERTIES OF AFG1 Based on chemical structure: Ketone groups in lactone ring can be reduced and produce cathodic wave. Unsaturated terminal furan ring can be reduced and produce cathodic wave. O

O

O

O

O O

O

Can forms anionic radical

Proposed mechanisme of the reduction of AFG1 that may take place at mercury electrode as reported by Smyth et al. (1979);

O

O

O

2

O O

2e-

O

ΘO

O

O

O O

O

2H+

O

O

2H2O

Dimer + 2OH-

UV-VIS SPECTRUM OF AFB1

BRb pH 6.0

BRb pH 9.0

BRb pH 7.0

BRb pH 11.0

UV-VIS SPECTRUM OF AFB2

BRb pH 6.0

BRb pH 7.0

BRb pH 9.0

BRb pH 11.0

UV-VIS SPECTRUM OF AFG1

BRb pH 6.0

BRb pH 9.0

BRb pH 7.0

BRb pH 11.0

UV-VIS SPECTRUM OF AFG2

BRb pH 6.0

BRb pH 9.0

BRb pH 7.0

BRb pH 11.0

ORAL PRESENTED AT SKAM-18 (JOHOR BAHRU, JOHOR ; 12 -14th SEPTEMBER 2005)

SQUARE-WAVE STRIPPING VOLTAMMETRIC TECHNIQUE FOR THE DETERMINATION OF AFLATOXIN B1 IN GROUNDNUT SAMPLES BY MOHAMAD HADZRI YAACOB AP DR ABDULL RAHIM MOHD YUSOFF PROF RAHMALAN AHAMAD (UTM)

OBJECTIVE OF THIS STUDY TO DEVELOP SWSV TECHNIQUE FOR THE DETERMINATION OF AFB1

INTRODUCTION • AFLATOXINS: Mycotoxin group Produced by two fungi; Aspergillus flavus and Aspergillus parasiticus

Types of aflatoxins:

AFB1 AFB2 AFG1 AFG2

AFM1 AFM2

Raw peanuts, peanuts products, wheat, maize, vegetable oils, dried fruites, barley, pear, cocoa, coffee, herbs, spices, wine, cottonseeds. Cow milk and its products, eggs and meat product

Carcinogenic, teratogenic and mutagenic IARC (1988): Class I carcinogenic materials

Regulatory level in Malaysia: Groundnuts < 15 ppb Milk < 0.5 ppb Others < 5 ppb

Chemical structure of AFB1 O O

O

O

O O

(2,3,6a,9a-tetrahydro-4-methoxycyclo penta[c] furo [3’,2’:4,5] furo [2,3-h][1] benzopyran-1,11-dione)

Produces blue colour under UV light MW = 312

EXPERIMENTAL INSTRUMENTS; VA 757 Metrohm Voltammetry analyser + MME Stand Working electrode = HMDE Ref electrode: Ag/AgCl, 3M KCl

REAGENTS; a) Britton-Robinson buffer(BRB) pH = 9.0 and AFB1 std b) Methanol, 0.1 N HCl, 15% ZnSO4, diatomoceus earth powder, chloroform (for extraction and clean-up) SAMPLES: Groundnut from different shops

EXPERIMENTAL FLOW CHART 1) Scan a blank

2) Scan AFB1 std

10 ML BRB SOLUTION

NITROGEN GAS IS FLOWED IN AFLATOXINS SOLUTION, REDISSOLVED IN BRB SOLUTION

SPIKE INTO 20 ML VOLTAMMETRIC CELL

SOLUTION IS DEGASSED

BLANK SOLUTION BLANK VOLTAMMOGRAM

SOLUTION IS SCANNED OBTAINED VOLTAMMOGRAM

BLANK SOLUTION + AFB1 SAMPEL VOLTAMMOGRAM

Method optimisation; Study effect of pH of BRB (6.0 to 13.0) potential window (Ei and Ef ) (0.4 to -1.2 V) Accumulation potential (Eacc) (0 to -1.4 V) Accumulation time (tacc) (0 to 150 s) Frequency (25 to 125 Hz) Voltage step (0.01 to 0.04 V) Amplitude (0.025 to 0.1 V) Scan rate

to peak height (Ip) and peak potential (Ep) of AFB1

RESULTS AND DISCUSSION Initial parameters (from optimum DPCSV parameters):

Ei = -1.0 V, Ef = -1.4 V, Eacc = -0.6 V, tacc = 80 s, scan rate = 50 mV/s and pulse amplitude = 80 mV. Supporting electrolyte = BRB pH 9.0. [AFB1] = 0.1 uM

Single cathodic peak Ep = -1.23 V Ip =

230 nA

Compare to DPCSV; Ep = -1.24 V; Ip = 60 nA

a) Effect of pH of BRB (use 0.1 uM AFB1) 300 200

Ep (-V)

I p (n A )

250 150 100 50 0 5

6

7

8

9

10

11

12

13

14

pH of BRB

1.34 1.32 1.3 1.28 1.26 1.24 1.22 1.2 1.18 5

6

7

8

9

10

pH

1. pH 9.0 gave maximum Ip 2. Ep shifted towards negative direction at higher pH Î Hydrogen ion involves in reduction process

11

Proposed mechanism; a) pH 6-8

O

O

O

O

H

H

O

O

2e

-

2H + OCH 3

OCH 3

b) pH 9-12 O

O

O

O

O

O

2e-

2

2

OCH3

OCH3

2 H 2O

(Ref: Smyth et al. (1979): used DPP technique)

D im e r

+ 2 OH-

b) Effect of Ei 300

I p (n A )

250 200 150 100 50 0 0.2

0.4

0.6

0.8

1

1.2

1.4

Ei (-V)

Ei = -1.0 V gave highest Ip ( 285 nA) Î optimum Ei for further experiments

BRB pH 9.0 Ei = -1.0 V

c) Effect of Eacc 400 350

I p (n A)

300 250 200 150 100 50 0 0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Eacc (-V)

BRB pH 9.0 Eacc = - 0.8 V gave highest Ip (366 nA). Î Eacc - 0.8 V was selected as optimum Eacc

Ei= -1.0 V Eacc = -0.8 V

d) Effect of tacc 450 400 350 I p (nA)

300 250 200 150 100 50 0 0

40

80

120

160

200

Tacc (s)

100 s is optimum tacc. It gave maximum Ip (400 nA)

BRB pH 9.0 Ei= -1.0 V Eacc = -0.8 V tacc = 100 s

e) Effect of frequency 1000

Ip (nA)

800 600 400 200 0 0

25

50

75

100

125

150

Frequency (Hz)

Frequency = 125 gave maximum Ip(820 nA). > 125 Hz, not possible due to instrument limitation. Too fast.

BRB pH 9.0 Ei= -1.0 V Eacc = -0.8 V tacc = 100 s Freq = 125 Hz

Ip is depend linearly on the square root of frequency ( 5 to 125 Hz); R 2 = 0.9919, n = 5.

y = 107.91x - 424.7

Î AFB1 absorbed on the mercury electrode 1000

I p (nA)

800 600 400 y = 107.91x - 424.7

200

R2 = 0.9919

0 3

5

7 (Frequency)1/2

9

11

f) Effect of voltage step 1200

I p (nA)

1000 800 600 400 200 0 0

0.01

0.02

0.03

0.04

0.05

Voltage step (V)

Voltage step = 0.03 V which gave maximum Ip (956 nA) was selected as the optimum v.step

BRB pH 9.0 Ei= -1.0 V Eacc = -0.8 V tacc = 100 s Freq = 125 Hz V.Step = 0.03 V

g) Effect of pulse amplitude 1200

I p (nA)

1000 800 600 400 200 0 0

0.025

0.05

0.075

0.1

0.125

Amplitude (V)

Amplitude = 0.05 V gave maximum Ip (956 nA)

BRB pH 9.0 Ei= -1.0 V Eacc = -0.8 V tacc = 100 s Freq = 125 Hz V.Step = 0.03 V Amplitude = 0.05 V

Square-wave frequency, voltage step and scan rate are interrelated; For frequency = 125 Hz and voltage step = 0.030 V Î scan rate = 3750 mV/s

Optimum parameters for the SWSV determination of AFB1 BRB pH 9.0, Ei = -1.0 V, Ef = -1.4 V, Eacc = -0.8 V, tacc = 100 s, Freq = 125 Hz, V.Step = 0.03 V, Amplitude = 0.05 V and scan rate = 3750 mV/s

Optimum SWSV parameters Ip= 956 nA (Ep = -1.30 V)

(b)

Non-optimum SWSV Ip= 230 nA (Ep= 1.23 V) Increases 4 times Compare to optimum DPCSV parameters Ip= 60 nA ( Ep= -1.24 V). Increases 16 times. ÎSWSV more sensitive and faster (scan rate = 3750 mV/s) compare to DPCSV. (* For DPCSV: 50 mV/s)

(a)

(a) DPCSV (b) SWSV

Calibration curve for AFB1 and method validation 1200

I p (n A )

1000 800 600 y = 70.97x + 76.588 R2 = 0.9984

400 200 0 0

5

10

15

20

[AFB1] x 10-8 M

Linearity range: 0.01 µM to 0.15 µM / 3.12 ppb to 46.8 ppb AFB1 LOD; 0.125 x 10-8 M / 0.39 ppb / 390 ppt * By DPCSV; LOD: 0.5 x 10-8 M / 1.56 ppb.

Precision / repetibility;(0.1 µM / 31.2 ppb) Ip ± SD / nA (RSD)

Ep (V)

Day 1:

932 ± 7.92 (0.85%)

-1.30

Day 2:

933 ± 7.74 (0.83%)

-1.30

Day 3:

933 ± 7.84 (0.84%)

-1.30

(n =5)

SWSV voltammograms of AFB1 (n=5): Day 1

Accuracy / Recovery: 0.05 uM:

99.67 ± 0.48% (n=3)

-1.30 V

0.10 uM:

100.47 ± 0.34% (n=3)

-1.30 V

(a)

(b)

Voltammograms of (a) 0.05 and (b) 0.10 uM AFB1 (n=3)

Analysis of AFB1 in groundnut sample 1) Extracted and cleaned-up: follows Chemistry Dept, MOSTI procedure. 2) Recovery studies – AFB1 in groundnut eluate Result; Amount added / ppb

Amount found /ppb

Recovery (%), n=3

3.00

2.82 ± 0.02

94.00 ± 0.67

9.00

8.21 ± 0.14

91.22 ± 1.56

15.00

13.88 ± 0.30

92.53 ± 2.00

Recovery: > 90 % is considered good recovery.

FLOW CHART OF SAMPLE ANALYSIS EXTRACTION SAMPLE

AND CLEAN UP (FOLLOW CHEM DEPT, MOSTI PROCEDURE)

INJECT 200 ul SAMPLE INTO CELL

PREPARE FINAL SOLUTION IN BRB pH = 9.0

(+10 Ml BRB pH = 9.0)

SCAN USING SWSV TECHNIQUE

STD ADDITION OF 10 ppb OF AFB1

RESCAN USING SWSV TECHNIQUE

Voltammogram of real sample + 10 ppb AFB1, compare with HPLC result

(c)

(b)

(a)

(a) Blank (b) 200 ul real sample (c) real sample + 10 ppb afb1

SWSV; 36.30 ppb

HPLC; 36.00 ppb

Voltammogram of real sample + 10 ppb afb1 compare with HPLC result

(a) Blank (b) 200 ul real sample (c) real sample + 10 ppb afb1 SWSV; ND

HPLC; ND

No peak for AFB1: Not detected.

Results; AFB1 in groundnut samples (duplicate analysis) No of sample

By SWSV/ ppb

By HPLC/ppb

1

ND

ND

2

ND

ND

3

ND

ND

4

9.21

8.25

5

13.92

14.34

6

36.30

36.00

t test: No significant different between both techniques (tcal = 0.51 < tcri (p : 0.05) = 2.57)

CONCLUSION SWSV technique for the determination of AFB1 in groundnut sample was successfully developed. The technique is precise, accurate, high sensitive, fast and low cost technique.

ACKNOWLEDGEMENT SUPERVISORS: AP DR ABDUL RAHIM MOHD YUSOFF PROF RAHMALAN AHAMAD

SHORT TERM GRANT: 75152/2004 SCHOOL OF HEALTH SCIENCES, USM , HEALTH CAMPUS, KUBANG KRIAN, KELANTAN

MR RADWAN, CHEM DEPT. MOSTI, PENANG BRANCH.

CV result for 1.3 uM AFB1 in BRB pH 9.0

Single cathodic peak at Ep = -1.310 V Î Irreversible reduction reaction at mercury electrode

FORMULA FOR CALCULATION AMOUNT OF AFB1 IN GROUNDNUT SAMPLE;

ppb aflatoxin = P’/ P x C x *12.5 ( for injection vol = 200 ul) ( P’ = Ip of sample (nA), P = Ip of std (nA) C = conc of injected std, 10 ppb)

*For other injection volume; 100 ul = x 25

300 ul = x 8.33

400 ul = x 6.25

500 ul = x 5

Voltammogram of real sample+ 10 ppb afb1, compare with HPLC result (c)

(b)

(a)

(a) Blank (b) 200 ul real sample (c) real sample + 10 ppb afb1

SWSV; 36.30 ppb Afb1 (ppb) = 94.3 x 10 x 12.5 = 36.30 ppb 419 – 94.3

HPLC; 36.00 ppb

gassed with

Ep of AFB2 chang n rincreasing 0.9 e scan ra 0.7 w p a ÎReduction of AF 0.5 iirriversible 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 e r reactio log scan rate t d h g a 4.3) Ip vs V r ta h L p 70 m e 60 hi en 50 rn 40 ( e c u 30 Ra 2 u m 20 ÎAFB2 was adso r lo

1.1

y = 0.5097x + 0.4115 R2 = 0.995

Ip (n A )

ices, herbs, duces blue hanol : 360 ctor.

CTION

4.

oxin, dangerous Type I carcinogen ctures; O

O

O

O

O

a) DPCSV voltammogr

O

O

O

O

ip = 61.71 nA OCH3

AFG1

O

OCH3

AFG2

ip = 59.97n

b) SQWSV voltammogr

: measures s applied. Simple, y and low cost

RIMENT

46 processor and de electrode (MME)

ip = 833 nA

ip = 816nA

c) DPCSV and SQWSV

Paper will be published in

Malaysian Journal of Analytical Science

1

SQUARE WAVE CATHODIC STRIPPING VOLTAMMETRIC TECHNIQUE FOR DETERMINATION OF AFLATOXIN B1 IN GROUND NUT SAMPLE Mohamad Hadzri Yaacob1, Abdull Rahim Hj. Mohd. Yusoff2 and Rahmalan Ahamad2 School of Health Sciences, USM, 16150 Kubang Krian, Kelantan, Malaysia Chemistry Dept., Faculty of Sciences, UTM, 81310 Skudai, Johor, Malaysia

1 2

Abstract An electroanalytical method has been developed for the detection and determination of the 2,3,6a,9a-tetrahydro-4-methoxycyclo penta[c] furo[3’,2’:4,5] furo [2,3-h][l] benzopyran-1,11-dione (aflatoxin B1, AFB1) by a square wave cathodic stripping voltammetric (SWSV) technique on a hanging mercury drop electrode (HMDE) in aqueous solution with Britton-Robinson Buffer (BRB) at pH 9.0 as the supporting electrolyte. Effect of instrumental parameters such as accumulation potential (Eacc), accumulation time (tacc), scan rate (v), square wave frequency, step potential and pulse amplitude were examined. The best condition were found to be Eacc of -0.8 V, tacc of 100 s, v of 3750 mV/s, frequency of 125 Hz, voltage step of 30 mV and pulse amplitude of 50 mV. Calibration curve is linear in the range of 0.01 to 0.15 uM with a detection limit of 0.125 x 10-8 M. Relative standard deviation for a replicate measurements of AFB1 (n = 5) with a concentration of 0.01 uM was 0.83% with a peak potential of -1.30 V (against Ag/AgCl). The recovery values obtained in spiked ground nut elute sample were 94.00 +/0.67 % for 3.0 ppb, 91.22 +/- 1.56 % for 9 ppb and 92.56 +/- 2.00 % for 15.0 ppb of AFB1. The method was applied for determination of the AFB1 in ground nut samples after extraction and clean-up steps. The results were compared with that obtained by high performance liquid chromatography (HPLC) technique. Abstrak Satu kaedah elektroanalisis telah dibangunkan untuk mengesan dan menentukan 2,3,6a,9atetrahydro-4-methoxycyclo penta[c] furo[3’,2’:4,5] furo [2,3-h][l] benzopyran-1,11-dione (aflatoxin B1, AFB1) menggunakan teknik voltammetri perlucutan katodik denyut pembeza di atas elektrod titisan raksa tergantung (HMDE) di dalam larutan akuas dengan larutan penimbal Britton-Robinson (BRB) pada pH 9.0 bertindak sebagai larutan penyokong. Kesan parameter peralatan seperti keupayaan pengumpulan (Eacc), masa pengumpulan (tacc), kadar imbasan (v), frekuensi gelombang bersegi, kenaikan keupayaan dan amplitud denyut telah dikaji. Keadaan terbaik yang diperolehi adalah Eacc; -0.8 V, tacc; 100 s, v; 3750 mV/s, frekuensi; 125 Hz, kenaikan keupayaan; 30 mV dan amplitud denyut; 50 mV. Keluk kalibrasi adalah linear pada julat di antara 0.01 ke 0.15 uM dengan had pengesan pada 0.125 x 10-8 M. Sisihan piawai relatif untuk 5 kali pengukuran AFB1 dengan kepekatan 0.01uM ialah 0.83 %. Nilai perolehan semula di dalam larutan elusi sampel kacang yang disuntik dengan 3.0 ppb, 9 ppb dan 15.0 ppb AFB1 adalah 94.00 +/- 0.67 %, 91.22 +/- 1.56 % dan 92.56 +/- 2.00 % masing-masingnya. Kaedah ini telah digunakan untuk menentukan kandungan AFB1 di dalam sampel kacang tanah selepas proses pengekstraksian dan pembersihan dijalankan. Keputusan yang diperolehi telah dibanding dengan keputusan dari kaedah kromatografi cecair berprestasi tinggi. Keywords: square wave stripping voltammetry, HMDE, aflatoxin B1, ground nut Introduction Aflatoxins (AF), the mycotoxin produced mainly by Aspergillus flavus and parasiticus and display strong carcinogenicity [1]. They are dangerous food and contaminants and represent a worldwide threat to public health. AFB1, B2, G1 and G2 and their metabolites M1 and M2 are the most common, 1

Corresponding address: Chemistry Dept, Faculty of Science, UTM, 81310 Skudai, Johor, Malaysia tel: 07-553 4492

2

and of these, AFB1 and AFG1 are observed most frequently in food [2]. Of these, researches have shown AFB1 (Fig 1) exhibits most toxic [3] with the order of toxicity is AFB1 > AFG1 > AFB2 > AFG2 indicates that the terminal furan moiety of AFB1 is a critical point for determining the degree of biological activity of this group of mycotoxins [4]. Many countries including Malaysia have stringent regulatory demands on the level of aflatoxins permitted in imported and traded commodities. O O

O

O

O O

Figure 1 Chemical structure of AFB1 One of the foodstuffs which is most occurrence of AFB1 is ground nut. In Malaysia, the AFB1 level in peanut is regulated with maximum level that cannot be greater than 15 ppb [5]. Several analytical techniques for quantitative determination of the AFB1 in ground nut have been proposed such as thin layer chromatograhpy [6], high performance liquid chromatography[ 7,8,9 ]and an enzyme-linked immunosorbent assay, ELISA [10], All these methods, however, require specialist equipment operated by skilled personel and expensive instruments and high maintenance cost [11]. Due to all these reasons, a voltammetric technique which is fast, accurate and require low cost equipment [12,13] is proposed. A square wave cathodic stripping which is presented in this paper is one of the voltammetric technique that in particularly, has a few advantages compared to other voltammetric technique such as high speed, increased analytical sensitivity and relative insensitivity to the presence of dissolved oxygen [14]. Previous experiment using cyclic voltammetric technique showed that AFB1 reduced at mercury electrode and the reaction is totally irreversible [15]. This work aimed to study and develop a SWSV method for determination of AFB1 at trace levels and to determine this aflatoxin in ground nut samples. No such report has been published regarding this experiment until now. Experiment Apparatus Square-wave voltammograms were obtained with Metrohm 693 VA Processor coupled with a Metrohm 694 VA stand. Three electrode system consisted of a hanging mercury drop electrode (HMDE) was used as the working electrode, Ag/AgCl/3 M KCl reference electrode and a platinum wire auxiliary electrode. A 20 ml capacity measuring cell was used for placing supporting electrolyte and sample analytes. All measurements were carried out at room temperature. All pH measurements were made with Cyberscan pH meter, calibrated with standard buffers at room temperature. Reagents AFB1 standard (1mg per bottle) was purchased from Sigma Co. and was used without further purification. Stock solution (10 ppm or 3.21 x 10-5 M) in benzene:acetonitrile (98:2) were prepared and stored in the dark at 14 0 C. The diluted solution were prepared daily by using certain volume of stock solution, degassed by nitrogen until dryness and redissolved in Britton-Robinson buffer (BRB) solution at

3

pH 9.0. Britton-Robinson buffer solutions (prepared from a stock solution 0.04 M phosphoric (Merck), boric (Merck) and acetic (Merck) acids; and by adding sodium hydroxide (Merck) 1.0 M up to pH of 9.0. All solutions were prepared in double distilled dionised water (~ 18M Ω cm). All chemicals were of analytical grade reagents. Procedure For voltammetric experiments, 10 ml of Britton-Robinson buffer solution with pH 9.0 was placed in a voltammetric cell, through which a nitrogen stream was passed for 600 s before recording the voltammogram. The selected Eacc = - 800 mV was applied during the tacc = 100 s while the solution was kept under stirring. After the accumulation time had elapsed, stirring was stopped and the selected accumulation potential was kept on mercury drop for a rest time (tr = 10 s), after which a potential scan was performed between -1.00 as initial potential (Ei) and finished at -1.400 V as final potential (Ef ) by SWSV technique. Procedure for the determination in ground nut samples AFB1 was extracted according to the standard procedure developed by Chemistry Department, Penang Branch, Ministry Of Science, Technology and Innovation, Malaysia [16]. 1ml of the final solution from extraction and clean-up steps in chloroform was pipette into amber bottle, degassed with nitrogen and redissolved in 1ml of BRB solution. 200 ul of this solution was spiked into 10 ml supporting electrolyte in volumetric cell. After that, the general procedure was applied and voltammogram of sample was recorded. This experiment was followed by a standard addition of 10 ppb of AFB1 standard addition and voltammogram of sample with AFB1 standard was recorded. Result and discussion Using previous differential pulse stripping voltammetry (DPCSV) optimum parameters [17], SWSV was run to determine 0.1 uM AFB1 in BRB pH 9.0. It gave a single reduction signal with peak height (Ip) of 250 nA at peak potential (Ep) of -1.26 V (against Ag/AgCl). The SWS voltammograms shows improved Ip compared to that obtained bt DPCSV where the Ip was increased almost 4 times as shown in Figure 2. Cyclic voltammogram of AFB1 in BRB pH 9.0 is shown in Figure 3 which only produced a cathodic peak that indicates the non-reversibility of the electrode process [18].

Figure 2 Voltammograms of 0.1 uM AFB1 obtained by (a) DPCSV and (b) SWSV techniques. Experimental condition; for DPCSV: Ei = -1.0 V, Ef = -1.4 V, Eacc = -0.6 V, tacc = 80 s, υ = 50 mV/s and

4

pulse amplitude = 80 mV and for SWSV: Ei = -1.0 V, Ef = -1.4 V, Eacc = -0.6 V, tacc = 80 s, frequency = 50 Hz, voltage step = 0.02, amplitude = 50 mV and υ = 1000 mV/s.

Figure 3 Cyclic voltammogram of AFB1 in BRB pH 9.0. Experimental conditions: V, Ehigh = 0 and scan rate = 200 mV/s.

Ei = 0, Elow = -1.5

The effect of the pH of the BRB on the stripping was studied in a pH range of 6 – 13 (Figure 4). The Ip increased slowly with increasing pH up to 8.0 followed with sharply increased for pH 8.0 to 9.0, then decreased in pH 10.0 and continuously decreased in a range of 10 – 13. Thus pH 9.0 was chosen for the analysis. This result is not contradicted with that found by Smyth et al. (1979) when they performed polarographic study of AFB1 [19].

300

I p (nA)

250 200 150 100 50 0 5

6

7

8

9

10

11

12

13

14

pH of BRB

Figure 4 Influence of pH of BRB on the Ip of 0.10 uM AFB1 using SWSV technique. The instrumental parameters are the same as in Figure 2.0. Figure 5 shows a dependence of the Ep on pH. Shifting of the Ep towards the negative direction at higher pH implies that the reduction process takes up hydrogen ions [20]. A double bond in aromatic ring conjugates with ketone group, in general, undergoes a reduction at mercury electrode. The suggested mechanism of this reaction in BRB pH 9.0 is illustrated in Figure 6 as reported by Smyth et al. (1979) [21].

5

Ep (-V)

1.34 1.32 1.3 1.28 1.26 1.24 1.22 1.2 1.18 5

6

7

8

9

10

11

pH

Figure 5

Relationship between Ep of AFB1 with pH of BRB

O

O

O

O

O

O

2e2H +

2

2 CH3

CH3

2 H 2O

D im e r

Figure 6

+ 2 OH-

Mechanism for reduction of AFB1 at mercury electrode in BRB pH 9.0

Optimisation of condition for the stripping analysis The voltammetric determination of analytes at trace levels normally involves very small current response. For that reason it is important to optimise all those parameters which may have an influence on the measured current. The effect of Ei , Eacc , tacc, frequency, voltage step and amplitude were studied. Square-wave voltammetric technique was used with stirring. For this study, 1.0 x 10-7 M of AFB1 was spiked into supporting electrolyte. A study of the influence of Ei showing a peak height (Ip) was obtained for Ei = -1.0 V (Figure 7). The Ip was slowly decreased for Ei more negative than -1.0 V. This value was chosen for subsequent studies further optimisation step. Influence of Eacc to Ip of AFB1 was investigated where the Eacc was varied between 0 to -1.4 V. The maximum value of Ip obtained at -0.8 V (366 nA) as shown in Figure 8. This value was selected for subsequent experiments.

6

The dependence of Ip on tacc was studied. The effect of tacc on the Ip was studied where tacc was varied from 0 to 160 s. The result is shown in Figure 9 which reveals that there is linearly up to 100s according to equation y = 3.8557x + 21.383 (n=6) with R2 = 0.9936, then it increased rather slowly leveling off at about 140 s. At 160 s, Ip decreased which may be due to the electrode saturation [21]. Thus, 100 s was appeared to be an optimum tacc for the pre-concentration prior to stripping.

300

Ip (nA)

250 200 150 100 50 0 0.2

0.4

0.6

0.8

1

1.2

1.4

Ei (-V)

Figure 7 Effect of Ei on the Ip of 0.1 uM AFB1 in BRB pH 9.0

400 350 300 Ip (nA)

250 200 150 100 50 0 0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Eacc (-V)

Figure 8 Effect of Eacc on the Ip of 0.1 uM AFB1 in BRB pH 9.0

450 400 350 Ip (nA)

300 250 200 150 100 50 0 0

40

80

120

160

200

T acc (s)

Figure 9 Effect of tacc on the Ip of 0.1 uM AFB1 in BRB pH 9.0

7

For other instrumental condition such as square wave frequency, step potential and pulse amplitude were examined, varying one of them and maintaining constant others. The variable ranges were: 25 to 125 Hz for the frequency, 0.01 to 0.04 for the voltage step and 25 – 100 mV for pulse amplitude. Generally, the Ip increase by increasing all of these instrumental parameters [22]. At higher potential values the peak width increase; at higher frequency values the current background increases. Finally, the condition selected were: frequency = 125 Hz, voltage step = 0.03 V and pulse amplitude = 50 mV (Figures 10 to 12). Under optimised parameters, Ip of AFB1 was 956 nA which is 16 times higher compared to that obtained by DPCSV. Figure 13 shows voltammograms of 0.1 uM AFB1 obtained by SWSV and DPCSV techniques. 1000

Ip (nA)

800 600 400 200 0 0

25

50

75

100

125

150

Fre que ncy (Hz)

Figure 10 Dependence of the Ip of AFB1 on SWSV frequency

1200

Ip (nA)

1000 800 600 400 200 0 0

0.01

0.02

0.03

0.04

0.05

Voltage step (V)

Figure 11 Dependence of the Ip of AFB1 on SWSV voltage step

8

1200

Ip (nA)

1000 800 600 400 200 0 0

0.025

0.05

0.075

0.1

0.125

Amplitude (V)

Figure 12 Dependence of the Ip of AFB1 on SWSV pulse amplitude

Figure 13 Voltammograms of AFB1 obtained by (a) DPCSV and (b) SWSV techniques in BRB pH 9.0. Voltammetric determination of AFB1 and analytical characteristics of the method Using the selected conditions already mentioned, a study was made of the relationship between Ip and concentration. There is a linear relationship in the concentration range 0.01 to 0.15 uM as shown in Figure 14. Limit of detection (LOD) was 0.389 ppb (0.125 x 10-8 M) which was determined by standard addition of low concentration of AFB1 until obtaining the sample response that is significantly difference from blank [23]. Relative standard deviation (RSD) of the analytical signals at several measurement (n=5) of 0.10 uM was 0.83%. 1400 1200

Ip(nA )

1000 800 600 y = 62.373x + 246.15

400

R2 = 0.9956

200 0 0

5

10

15

20

[AFB1] / 10-8 M

Figure 14 Calibration plot of AFB1 in BRB pH 9.0 obtained by SWSV technique.

9

Determination of AFB1 in ground nut samples The proposed method was applied to the analysis of the AFB1 in ground nut samples. Recovery studies were performed by spiked with difference concentration levels of AFB1 standard into eluate of ground nut sample. In this case the concentrations used were 3 ppb (0.963 x 10-8 M), 9 ppb (2.889 x 10-8 M) and 15 ppb (4.815 x 10-8 M). The results of these studies are shown in Table 1. For analysis of AFB1 in ground nut samples, the standard addition method was used in order to eliminate the matrix effects. Figure 15 show voltammograms of real sample together with spiked AFB1 standard. Table 2 listed the content of AFB1 in 6 samples obtained by proposed technique compared with that obtained by HPLC. The results show that there are no significant different of AFB1 content obtained by both techniques.

Table 1

Percent recovery of AFB1 spiked in real samples (n=3)

Amount added (ppb)

Amount found (ppb)

Recovery (%) n=3

3.00

2.82 +/- 0.02

94.00 +/- 0.67

9.00

8.21 +/- 0.14

91.22 +/- 1.56

15.00

13.88 +/- 0.30

92.53 +/- 2.00

Figure 15 SWS voltammograms of real sample (b) and spiked AFB1 (c) obtained in BRB pH 9.0 as the supporting electrolyte (a)

10

Table 2 AFB1 content in ground nut samples by proposed technique compared with those obtained by HPLC.

No of sample

AFB1 content in real sample

By SWSV

By HPLC

1

ND

ND

2

ND

ND

3

ND

ND

4

9.21

8.25

5

13.92

14.34

6

36.30

36.00

Conclusion A SWSV technique was successfully developed for the determination of AFB1 in ground nut as an alternative method for determination of AFB1 which is sensitive, accurate and fast technique. The results are not significant different with that obtained by accepted technique used for routine analysis of AFB1. Acknowledgement One of the author would like to thank University Science Malaysia for approving his study leave and financial support to carry out his further study at Chemistry Dept., UTM. We also indebt to UTM for short term grant (Vot No 75152/2004) and to Chemistry Department, Penang Branch, Ministry of Science, Technology and Innovation (MOSTI) for their help in analysis of aflatoxin in ground nut samples using HPLC technique. References 1.

Akiyama, H. Goda, Y. Tanaka, T and Toyoda, M. (2001). “Determination of aflatoxinsB1, B2, G1 and G2 in spices using a multifunctional column clean-up” J. Of Chromatography A, 932. 153-157.

2.

Yoruglu, T, Oruc, H.H. and Tayar, M. (2005) “Aflatooxin M1 levels in cheese samples from some provinces of Turkey” Food Control. 16(10): 883-895.

3.

World Health Organisation in: IARC Monograph on the evaluation of carcinogenic risk to human, WHO, Lyon, 1987.

11

4.

Hall, A.J., Wild, C.P in: Eaton, D.L., Groopman, J.D. (Eds). “The toxicology of aflatoxins: Human Health, Veterrinary and Agricultural Significance” Academic, New York, 1994.

5.

Malaysian Food Act (1983) Act No 281, Reviewed 2002, Food Quality Control Dept, Ministry of Health, Malaysia.

6.

Bicking,M.K.L., Kniseley, R.N and Svec, H.J (1983) “Coupled-Column for quantitating low levels of aflatoxins” J . Assoc. Off. Anal. Chem. 66, 905-908.

7.

Beebe, B.M. (1978). “Reverse phase high pressure liquid chromatographic determination of aflatoxins in foods” J. Assoc. Off. Anal. Chem. 81. 1347-1352.

8.

Cepeda, A., Franco, C.M., Fente, C.A., Vazquez, B.I., Rodriguez, J.L., Prognon, P. and Mahuzier, G. (1996) “Postcolumn excitation of aflatoxins using cyclodextrins in liquid chromatography for food analysis” J Of Chrom A. 721. 69-74.

9.

Garner, R.C., Whattam, M.M. , Taylor, P.J.L. and Stow, M.W ( 1993) “Analysis of United Kingdom purchased spices for aflatoxins using immunoaffinity column clean up procedure followed up by high-performance liquid chromatographic analysis and post-column derivatization with pyridium bromide perbromide” J. Chromatography, 648. 485-490

10.

Beaver, R.W., James, M.A. and Lin ,T.Y. (1991). “ELISA-based screening test with liquid chromatography for the determination of aflatoxin in corn” J. Assoc.Off. Anal. Chem , 74: 827 – 829

11.

Garden, S.R. and Strachan, N.J.C.(2001). “Novel colorimetric immunoassay for the detection of aflatoxin B1” Anal Chim Acta, 444. 187-191.

12.

Braitina, Kh. Z., Malakhova, N.A. and Stojko, Y. (2000) “Stripping voltammetry in environmental and food analysis” Fresenius J Anal Chem 368. 307-325

13.

Skrzypek, S., Ciesielski, W., Sokolowski, A., Yilmaz, S. and Kazmierczak, D. (2005) “Square wave adsorption stripping voltammetric determination of famotidine in urine”Talanta. Article in press.

14.

Economou, A., Bolis, S.D., Efstathiou, C.E. and Volikakis, G.J (2002) “A virtual electrochemical instrument for square wave voltammetry”.Anal. Chim. Act. 467. 179-188.

15.

Yaacob, M.H., Mohd Yusoff, A.R. and Ahmad, R. (2003). “Cyclic voltammetry of AFB2 at the mercury electrode”. Paper presented at Simposium Kimia Malaysia ke 13 (SKAM-13), 9 – 11th September 2003, Kucing, Sarawak, Malaysia.

16.

Standard procedure for determination of aflatoxins (2000). Chemistry Dept. Penang Branch, Ministry of Science, Technology and Innovation (MOSTI), Malaysia – unpublished paper.

17.

Yaacob, M.H., Mohd Yusoff, A.R. and Ahamad, R. (2005). “Determination of the aflatoxin B1 in ground nut by differential pulse cathodic stripping voltammetry technique”. Paper presented at 2nd National Seminar On Chemistry, 14th April 2005, Universiti Sumatera Utara, Medan, Indonesia.

12

18.

Rodriguez, J., Berzas, J.J., Casteneda, G. and Rodriguez, N. (2005). “Voltammetric determination of Imatinib (Gleevec) and its main metabolite using square-wave and adsorptive stripping squarewave techniques in urine samples”. Talanta. 66. 202-209.

19.

Smyth, M. R., Lawellin, D. W. and Osteryoung, J.G. (1979). “Polarographic study of Aflatoxin B1, B2, G1 and G2: Application of differential pulse polarography to the determination of Aflatoxin B1 in various foodstuffs” Analyst. 104. 73-78

20.

Sun, N., Mo, W-M., Shen, Z-L. and Hu, B-X. (2005). “Adsorptive stripping voltammetric technique for the rapid determination of tobramycin on the hanging mercury electrode”. J Pharm. Biomed. Anal. Article in press.

21.

Wang J. (1994) “Analytical Electrochemistry” VCH Publisher, USA.

22.

Komorsky-Lovric, S., Lovric, M. and Branica, M. (1992). “Peak current-frequency relationship in adsorptive stripping square-wave voltammetry”. J of Electroanal. Chem. 335(1-2). 297-308.

23.

Barek, J. , Fogg, A. G., Muck, A. and Zima, J. (2001). “Polarography and Voltammetry at Mercury Electrodes”, Critical Reviews In Analytical Chemistry, 31. 291-309

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1

DEVELOPMENT OF DIFFERENTIAL PULSE STRIPPING VOLTAMMETRIC (DPSV) TECHNIQUE FOR DETERMINATION OF AFLATOXIN G1 AT THE MERCURY ELECTRODE Mohamad Hadzri Yaacob1, Abdull Rahim Hj. Mohd. Yusoff2 and Rahmalan Ahamad2 1

School of Health Sciences, USM, 16150 Kubang Krian, Kelantan

2

Chemistry Dept., Faculty Of Sciences, UTM, 81310 Skudai, Johor

Abstract DPSV technique was developed for determination of aflatoxin G1 (AFG1) in Britton-Robinson Buffer (BRB). Controlled Growth Mercury Electrode (CGME) and Ag/AgCl have been used as working and reference electrodes respectively. In this experiment, the analyte was initially scanned from -200 mV to -1500 mV in BRB from pH 2.0 to 9.0. The effect of pH of supporting electrolyte, scan rate, accumulation potential, accumulation time and pulse amplitude on the peak potential and peak current of the analyte were studied. The results showed that AFG1 compound undergoes reduction at potential -1175 mV ( versus Ag / AgCl ). Maximum peak height was observed in BRB pH 9.0 with the optimised parameters such as scan rate; 50 mV/s, accumulation potential; -600 mV, accumulation time; 80s and pulse amplitude; 80 mV . Under the optimum condition, the peak height of AFG1 was proportional to the concentrations of the AFG1 in the range of 0.02 to 0.26 uM with a limit of detection of 0.015 uM. Relative standard deviation for a replicate measurements of AFG1 ( n= 8) with the concentrations of 0.06 and 0.20 uM were 3.52% and 1.54% respectively. Recoveries of the different AFG1 concentrations in prepared standard solutions were found to be 85% to 95% with the precision around 0.4% to 1.0%.

1

Correspondence address; Chemistry Dept, Faculty Of Science, UTM 81310 Skudai Johor

2

Introduction

Aflatoxin G1 ( 3,4,7a,10a-tetrahydro-5-methoxy-1H, 12H furo[3’,2’:4,5]furo[2,3h]pyrano[3,4-c][1]-benzopyran-1,12-dione), structural formula is shown in Figure 1.0, is one of the type of aflatoxin which is naturally occurance (1). It is one of the compound in mycotoxin group (2) that is produced by Aspergillus flavus and Aspergillus parasticus fungi (3,4). It is found in various contaminated food such as peanuts and peanut products, barley, maize, cottonseed, coffee beans and others (5). It produces green color under ultraviolet light (6,7). In health aspect, it is considered as human carcinogen by the International Agency for Research on Cancer (IARC) as reported in World Health Organisation (WHO)’s monograph (8). It has been reported to be teratogenic, tremorogenic, and haemorrhagic to wide range of organisms and to cause hepatic carcinoma in man (9) Consequently, AFG1 level in animal feed and various human food is now monitored and tightly regulated by various countries. In this country, regulatory level for AFG1 and total aflatoxins is 15 ppb in raw peanuts and 5 ppb for others (10). Due to all these reasons, selecting appropriate and accurate method of analysis of AFG1 is absolutely necessary to determine AFG1 at the part-of-billion (ppb) level. O

O

O

O

O O

O

Figure 1.0 Chemical structure of AFG1 There have been several reports for the determination of AFG1 including thin layer chromatography (11,12), high performance liquid chromatography with various type of detectors ( 13,14,15, 16 ) and polarographic technique (17) but

3

no report has been published concerning the use of

stripping voltammetric

technique. The development of new method capable of determining AFG1 in food sample is important. Stripping voltammetric technique was selected for this study due to its simple, easy to use, sensitive and accurate technique (18). This technique applies pre-concentration step that will increase sensitivity of the method (19). This paper will describe the development of DPSV technique for determination of AFG1 using CGME as the working electrode.

Materials and Methods All reagents employed were of analytical grade. Water purified from a Nano Pure Ultrapure water system ( Branstead / Thermolyne) was used for all dilution and sample preparation. AFG1 was purchased from SIGMA: 1 mg was dissolved in hexane: acetonitrile

(98:2) to produce 10 ppm solution.

A stock of Britton-

Robinson buffer (BRB) solution 0.04 M was prepared as follows: 2.47 g boric acid (Fluka), 2.30 ml acetic acid (MERCK) and 2.70 ml orthophosphoric acid ( Ashland Chemical ) were dissolved in 1 l deionised water. The pH of the solution was adjusted to the desired values by adding 1 M sodium hydroxide (BDH) or 1 M hydrochloric acid ( MERCK).

Hexadistilled mercury, grade 9N, used for

working electrode was purchased from MERCK. All voltammograms were recorded with a BAS CV-50W Voltammetric Analyser in connection with a Controlled Growth Mercury Electrode (CGME) stand equipped with a three-electrode system consisting of an Ag / AgCl reference electrode, a platinum wire counter electrode and the CGME as working electrode. A 20 ml capacity BAS MR-1208 cell was used. In all voltammetric analysis, the supporting electrolyte solution was deaerated by a stream of nitrogen for at least 10 min. A pH meter Cyber-scan model equipped with a glass electrode combined with an Ag / AgCl reference electrode, was employed for the pH measurements. Differential pulse stripping voltammograms (DPSVs) of AFG1 were run after purging the test solution for at least 5 min. The DPSVs were scanned from -200 to -1500 mV with scan rate of 40 mV/s, accumulation potential ( Eacc ) -800 mV,

4

accumulation time ( tacc ) 40 s, pulse amplitude 50 mV, pulse width 50-ms and pulse period 200-ms.

Results and Discussion From our previous studies on the cyclic voltammograms of AFG1 in BRB pH 9.0, a reduction peak was observed at -1245 mV ( against Ag / AgCl) and no anodic peak at oxidation wave. The results of this CVs showed that AFG1 underwent irreversible reduction reaction at the mercury electrode (20).

Due to this

electroanalytical properties, differential pulse cathodic stripping voltammetric technique was applied in this study using the same supporting electrolyte. By using initial parameters as mentioned before, the peak potential ( Ep ) of 0.15 uM AFG1 was obtained at -1175 mV with peak current ( Ip ) of 2.0 ).

34.73 nA ( Figure

Several parameters have been optimised so as to obtain a more

symmetrical and higher reduction peak.

Figure 2.0 Voltammograms of 0.15 uM AFG1 (b) in BRb pH 9.0 (a) . Condition; scan rate; 40 mV/s, accumulation potential ( Eacc ); -800 mV, accumulation time ( tacc ); 40 s, pulse amplitude; 50 mV, pulse width; 50-ms and pulse period; 200ms.

Effect of pH of BRB The effect of different pH medium of BRB have been studied.

The results

showed that a reduction peak was first observed at pH 5.0 which increases in

5

peak height as the pH increases. It reaches its optimum height at pH 9.0. At pH more than 9.0, the peak height decreases rapidly and no peak was observed at pH more than 12.0 ( Fig 3.0 ). This results suggested that proton ion played a significant role in the reduction of AFG1 on mercury electrode. Hence, BRB with pH 9.0 was applied in this experiment..

50

Ip(nA)

40 30 20 10 0 4

6

8

10

12

pH of BRb

Figure 3.0 The effect of pH of BRB on peak current of AFG1. [AFG1] = 0.15 uM Condition: scan rate; 40 mV/s, accumulation potential ( Eacc ); -800 mV, accumulation time ( tacc ); 40 s, pulse amplitude; 50 mV, pulse width; 50-ms and pulse period; 200-ms.

Effect of potential windows, accumulation potential ( Eacc ) and accumulation time ( tacc) ) The influence of potential windows on the cathodic peak current of AFG1 was studied by varying the potential windows from -200 to -1000 mV and from -1400 to -1500 mV for initial ( Ei ) and final potential ( Ef ) respectively. The results showed that with Ei = -950 mV and Ef = -1400 mV, the peak current at Ep = 1160 mV was 41.50 nA which is higher than using initial potential windows as described in previous experiment. Therefore, this potential window was used for all subsequent measurements. The dependence of the cathodic peak current on Eacc was also studied. The highest peak current was found when Eacc was -600 mV and when Eacc changed to more negative than -600 mV, the Ip decreases

6

( Figure 4.0 ) which indicated that maximum accumulation of AFG1 at mercury electrode occurred when potential of -600 mV was applied on it in BRB pH 9.0. The effect of tacc on Ip of AFG1 also was studied. The Ip of AFG1 was found to increase with increasing tacc ( Figure 5.0 ) indicating continueous accumulation of AFG1 at the electrode surface with increasing accumulation time. Normally, the increase in the response current continued until a maximum signal level presumable corresponding to a saturation electrode surface was attained (21). The results thus obtained indicated that the optimum tacc for AFG1 was 80 s. Hence 80 s accumulation time was employed throughout this experiment.

I p (n A )

60 40 20 0 0

200

400

600

800

1000

1200

1400

1600

Eacc (-mV)

Figure 4.0 Effect of accumulation potential on Ip of AFG1

ip (n A )

80 60 40 20 0 0

50

100

150

200

tacc (s)

Figure 5.0 Effect of accumulation time on Ip of AFG1

7

Effect of instrumental parameters As the scan rate was varied from 20 to 100 mV s-1, the Ip increases and reached it maximum value at scan rate of

50 mV s-1 , after which the peaks became

broader with undesired tail at their end. A similar pattern for Ip was obtained with increasing pulse amplitude ( from 30 to 120 mV ). A pulse amplitude of 80 mV with scan rate of 50 mV s-1 were chosen for further studies. Using all optimised parameters ( Ei = -950 mV, Ef = 1400 mV, Eacc = -600 mV, tacc = 80 s, scan rate = 50 mV s-1, pulse amplitude = 80 mV ) the Ip of 0.15 uM AFG1 was 75.10 nA with Ep at -1150 mV was obtained Calibration graph, detection limit, precision and recovery

The

differential

pulse

cathodic

stripping

voltammograms

at

different

concentrations of AFG1 under optimum conditions are shown in Fig. 6.0. The Ip of AFG1 increased linearly with increasing concentration up to 0.26 uM. A linear calibration graph was obtained in the concentration range 0.02 to 0.26 uM AFG1 ( n = 10, r = 0.9980). At higher concentrations Ip tend to level off which may be due to electrode surface saturation (20). The detection limit ( three times signalto-noise ) was found to be 0.015 uM AFG1. For eight successive determinations of 0.06 and 0.20 uM AFG1, relative standard deviations were 3.52 and 1.54 % respectively which indicated that the precision of the method was good. Recovery of the different AFG1 concentrations in prepared standard solutions were found to be 85 to 95% with precision around 0.4 to 1.0 % as listed in Table 1.0.

8

Ip (n A )

160 140 120 100 80 60 40 20 0

y = 5.5958x + 3.7725 R2 = 0.998

0

0.05

0.1

0.15

0.2

0.25

0.3

[AFG1] / uM

(A)

(B)

Figure 6.0 (A) : Differential pulse cathodic stripping voltammograms of AFG1 at mercury electrode. AFG1 concentrations in (a) BRB pH 9.0; (b) 0.02 uM, (c) 0.04 uM, (d) 0.06 uM, (e) 0.08 uM, (f) 0.1 uM, (g) 0.12 uM, (h) 0.14 uM, (i) 0.18 uM, (j) 0.22 uM, (k) 0.26 uM. (B) The related calibration graph. Conditions: Ei = 950 mV, Ef = -1400 mV, Eacc = -600 mV, tacc = 80 s, scan rate = 50 mV s-1, pulse amplitude = 80 mV .

9

Table 1.0 Recovery of spiked AFG1 standard in BRB pH 9.0

[AFG1]

Ip (nA)

Ep (-mV)

injected

0.10 uM

[AFG1]

% recovered

obtained

56.35

1150

0.0940 uM

94.00

56.84

1150

0.0948 uM

94.80

56.62

1150

0.0942 uM

94.20 x = 94.50 +/- 0.41 (RSD = 0.43%)

0.15 uM

78.21

1140

0.1330 uM

88.68

77.19

1140

0.1313 uM

87.51

77.20

1140

0.1313 uM

87.51 x = 87.91 +/- 0.58 (RSD = 0.77%)

0.20 uM

98.42

1140

0.1691 uM

84.57

99.23

1140

0.1705 uM

85.75

98.14

1140

0.1686 uM

84.30 x = 84.87 +/- 0.77 (RSD = 0.91%

10

Conclusion

A differential pulse cathodic stripping voltammetric technique using mercury electrode as the working electrode for determination of AFG1 has been developed. The method presented here is fast, reliable and sensitive technique for analysis of AFG1 with good precision and recovery. Further study is currently been carried out for analytical application of this method for quantitation of AFG1 in food sample.

Acknowledgement The author gratefully acknowledge the University Science Malaysia for giving me a study leave together with a scholarship under the Academic Staff For Higher Education Scheme (ASHES). Also we would like to thank all Electroanalytical group and Chemistry Department staff, UTM for their fully cooperation.

References [1] Goldblatt L.A. 1969. Aflatoxin:Scientific background, control and implication. Academic Press, New York, pp 5-10 [2] Salleh B. 1998. Mikotoksin: Implikasinya terhadap kesihatan manusia dan haiwan, USM, Penang, pp 32 - 49 [3] Begum F and Samajpathi N. 2000. Mycotoxin production in rice, pulses and oilseeds, Naturwissenschaften, 87: 275-277. [4] Ordaz J.J., Fente C.C., Vazquez B.I., Franco C.M. and Cepeda A. 2003. Development of a method for direct visual determination of aflatoxin production by colonies of the Asperfillus flavus group, Int. J. Food Microbiology, 83, 219225. [5] Batista L. R., Chalfoun S.M., Prado G. Schwan R.F. and Wheals A. E. 2003. Toxigenic fungi associated with processed (green) coffee beans ( Coffea arabica L.) . Int. J. Food Microbiology, 85, 293-300.

11

[6] Akiyama H. Goda Y. Tanaka T and Toyoda M. 2001. Determination of aflatoxins B1, B2, G1 and G2 in spices using a multifunctional column clean-up, J. Of Chromatography A, 932. 153-157 [7] Aziz N.H., Youseff Y.A., El-Fouly M.Z. and Mousse L.A. 1998. Contamination of some common medicinal plant samples and spices by fungi and their mycotoxins. Bot. Bull. Acad. Sin 39, 279-285. [8] World Health Organisation 1987 in: IARC Monograph on the Evaluation of Carcinogenic Risk to Human, WHO, Lyon , pp 82 Supl. I [9] Refai M.K. 1988. Aflatoxins and aflatoxicosis. J. Egypt Vet. Med. Ass. 48, 119 [10] Ministry of Health, Malaysia, Food Act No 231. 1983. Reviewed 2003 [11] Shotwell O.L and Goulden M.L. 1977. Aflatoxins: Comparison of analysis of corn by various methods, J. Assoc. Off. Anal. Chem 60: 83-88 [12] Gimeno A. and Martins M. L. 1983 Rapid thin layer chromatographic determination of patulin, citrinin and aflatoxin in apples and pears and their juices and jams, J. Assoc. Anal. Chem, 66, 85-91. [13] Scholten J.M and Spanjer M.C. 1996. Determination of aflatoxin B1, B2, G1 and G2 in pastachio kernels and shells, , J. Assoc. Off. Anal. Chem 79: 13601364 [14] Jeffery H.W., Lenorich L.M and Martin R. Jr. 1982. Liquid chromatography determination of aflatoxins in artificially contaminated cocoa beans. , J. Assoc. Off. Anal. Chem 80: 1215-1219 [15] Joshua, H. 1993 Determination of aflatoxin by reversed-phase high performance liquid chromatography with post-column in-line photochemical derivatization and fluorescence detection, J. Chromatography A, 654: 247-254 [16] Gonzalez M.P.E., Mattusch J. and Wennrich R 1998. Stability and determination of aflatoxins by high-performance liquid chromatography with amperometric detection, J. Chromatography A, 828: 439-444 [17] Smyth M. R., Lawellin D. W. and Osteryoung J.G 1979. Polarographic Study of Aflatoxin B1, B2, G1 and G2: Application of Differential-pulse

12

Polarography to the Determination of Aflatoxin B1 in Various Foodstuffs, Analyst, 104: 73-78 [18] Braitina Kh. Z., Malakhova N.A. and Stojko Y. 2000 Stripping voltammetry in environmental and food analysis, Fresenius J Anal Chem 368: 307-325 [19] Fifield, F.W. and Haines, P.J. 2000. .Environmental Analytical Chemistry 2nd Ed, Balckwell Science UK, pp 241-242 [20] Yaacob M.H., Mohd Yusof A.R., Ahamad R. and Yakob A.R. 2004. Cyclic Voltammetry of AFG1 at mercury electrode, Paper will be presented at 3rd Annual Seminar on Sustainable Science and Management, KUSTEM, Terengganu, 4 – 5 May 2004. [21] Wang. 2000. Analytical Electrochemistry. Wiley-VCH, USA.

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DIFFERENTIAL PULSE STRIPPING VOLTAMMETRIC TECHNIQUE FOR THE DETERMINATION OF AFLATOXIN B2 AT THE MERCURY ELECTRODE

Mohamad Hadzri Yaacob, Abdull Rahim Hj. Mohd. Yusoff and Rahmalan Ahamad Chemistry Dept., Faculty Of Sciences, UTM, 81310 Skudai, Johor ABSTRACT Differential pulse voltammetric technique was developed for determination of aflatoxin B2 standard (AFB2) in Britton-Robinson Buffer (BRB). Hanging mercury drop electrode and Ag/AgCl have been used for working and reference electrodes respectively. In this experiment, the analyte was initially scanned from -1000 mV to -1500 mV in BRB pH 9.0. The effect of pH of supporting electrolyte, scan rate, accumulation potential, accumulation time and pulse amplitude on the peak potential and peak current of the analyte were observed. The results showed that AFB2 compound undergoes reduction reaction at a potential of -1230 mV ( versus Ag/AgCl ). BRB pH 9.0 was the best supporting electrolyte with the optimised parameters such as scan rate; 50 mV/s, accumulation potential; -800 mV, accumulation time; 80s and pulse amplitude; 80 mV which gave the maximum peak height. Under the optimum condition, the peak height of AFB2 was proportional to the concentrations of the AFB2 in the range of 0.02 to 0.32 uM with a limit of detection was 0.0159 uM. Relative standard deviation for a eplicate measurements of AFB2 ( n= 8) with the concentrations of 0.06 and 0.20 uM were 2.80% and 2.53% respectively. Recoveries of the different AFB2 concentrations in prepared standard solutions were found to be 85% to 95% with the precision around 0.7% to 2.0%. Keywords: aflatoxin B2 compound, differential pulse stripping voltammetry, Britton-Robinson buffer and hanging mercury drop electrode (HMDE)

1. INTRODUCTION Aflatoxins are mycotoxins produced by certain fungi, especially, Aspergillus flavus and Aspergillus parasiticus which are found on crops and foodstuffs under certain conditions (1). They display strong carcinogenicity. Therefore, they are dangerous food contaminants. They were first identified as the causative agent of the severe outbreak of “Turkey X” disease, a toxicosis that killed more than 100,000 turkey poults in England in 1960 (2) There are a major concern as human hepatocarcinogens and are considered to play an important role in the high incidence of human hepatocellular carcinoma in certain areas of the world. Due to this reason, many countries including Malaysia have set regulatory demands on the level of aflatoxins permitted in imported and trade commodities.

2

There are six main aflatoxin compounds such as Aflatoxin B1, B2, G1 and G2 ( designated as AFB1, AFB2, AFG1 and AFG2 ) and aflatoxin M1 and M2 ( designated as AFM1 and AFM2). The former four compounds are naturally prevalent and the others are hydroxylated metabolite of AFB1 and AFB2 and were found in the milk of lactating animals following ingestion of B1 and B2- contaminated feeds (3) Among them AFB1 exhibits potent carcinogenic and mutagenic characteristics in a numbers of human and animals. AFB2 has a similar chemical structure with AFB1 whereby the presence of an 8,9-double bond in the form of a vinyl ether at the terminal furan ring in AFB1, but not in AFB2. The chemical structure of AFB2 is shown in Figure 1.0. This small difference in structure is associated with very significant change in activity; whereby AFB1 is more toxic than AFB2 (4).

O O

O

O

O O

Figure 1.0: Chemical structure of AFB2 Due to their toxicity, it is important that the level of these contaminants be controlled in food, soils and water to prevent toxic effect. The way to achieve this is by monitor samples so that any problem can be identified and dealt with, by doing analysis of aflatoxins using the analytical techniques at the parts-per-billon (ppb) level. Current analysis is done by various methods including thin-layer chromatography (TLC) (5), liquid chromatography (LC) and high-performance liquid chromatography (6,7) and microtitre plate enzyme-linked immunosorbant assay (ELISA) (8). All these methods require special equipments operated by skilled personnel and time consume. With the aim of rapid, simple, accurate and relative low cost analysis, the voltammetric technique is proposed for this purposes (9). Voltammetric technique generally refer to a system that combines a triple electrodes system with a potentiostate for controlling applied potential. It measures the current response generated by mass transport of electroactive species in the solution to the working electrode while potential is scanned. The total current response is proportional to the amount of analyte present in the solution (10). This paper reports the differential pulse voltammetric determination of AFB2 at a mercury electrode using a cathodic stripping voltammetric technique. 3

2. EXPERIMENT 2.1

Reagents

All reagents employed were of analytical grade. Water purified from a Nano Pure Ultrapure water system ( Barnstead / Thermolyne) was used for all dilution and sample preparation. AFB2 was purchased from SIGMA ( 1 mg per bottle ) and dissolved in hexane:acetonitrile (98:2) produced 10 ppm standard solution. The stock solutions were placed in small amber glass tubes and kept in a freezer at -4.0oC. A stock solution of 0.04 M Britton-Robinson Buffer (BRB) solution was prepared as follows: 2.47 g boric acid (Fluka), 2.30 ml acetic acid (MERCK) and 2.70 ml orthoposphoric acid ( Ashland Chemical ) were diluted to 1 l with deionised water. Britton-Robinson Buffer of pH 2.0 to 13.0 were prepared by adding sodium hydroxide 1.0 M (MERCK) into the stock solution and was used as the supporting electrolyte. Hexadistilled mercury, grade 9 N, used in a control growth mercury electrode ( CGME ) stand, was purchased from MERCK. The purging was carried out with 99.99% nitrogen.

2.2

Instruments

A BAS CV-50W voltammetric analyser in connection with a CGME stand equipped with a three-electrode system and a 20 ml capacity BAS MR-1208 cell were used for all the voltammetric determination. The working electrode was a hanging mercury drop electrode ( HMDE ). A silver-silver chloride (Ag / AgCl ) and a platinum wire were used as the reference and counter-electrode respectively. BAS CV-50W was connected to a computer for data processing. A pH meter Cyber-scan model equipped with a glass electrode combined with an Ag/AgCl reference electrode, was used for all pH measurements.

2.3

Procedure

2.3.1 General procedure The general procedure used to obtain cathodic stripping voltammograms was following this procedure. A 10 ml aliquot of supporting electrolyte solution was put in a voltammographic cell. The stir was switched on and the solution was purged with nitrogen gas for 25 minutes. After forming a new HMDE, accumulate was effected for the required time at the appropriate potential while stirring the solution. At the end of the accumulation time the stirring was switched off and after 10 s had elapsed to allow the solution to become quiescent, the negative going potential was initiated. Ag/AgCl saturated was used as the reference electrode throughout this study.

4

2.3.2 Procedure for determination of aflatoxin For the aflatoxin stock standard solution, organic solvent was removed by flowing nitrogen to dryness followed by addition of the BRB supporting electrolyte solution. The solution was placed in small amber tube. This solution has been freshly prepared before voltammetric experiment started. When further volume of aflatoxin were added using a micropipette into the cell, the solution was redegassed with nitrogen for another 5 minutes before further voltammetric experiment was carried out.

3. RESULTS AND DISCUSSION 3.1 The effect of pH of supporting electrolyte

Ip (n A )

From our previous cyclic voltammetric studies of AFB2 compound, it was capable of undergoing reduction process at the mercury electrode in BRB pH 9.0 (11). In the present study, 2.0 uM AFB2 was cathodically scanned in BRB solution in the pH range of 3.0 to 13.0. This study was carried out with the aim to determine if protons are directly involved in the reduction process of AFB2. The result showed that in the BRB with pH < 4.0 , no peak was observed. The single and characteristic AFB2 peak was initially observed starting from pH 5.0 with Ip and Ep were 11.0 nA and -1119mV respectively. The effect of the pH of supporting electrolyte on the Ip of the AFB2 peak is shown in Figure 1.0

60 50 40 30 20 10 0 4

6

8

10

12

pH

Figure 1.0:

Dependence of the peak height of AFB2 on the pH of 0.04 M BRB solution. AFB2 concentration: 2.0 uM, Ei = 0, Ef = -1.5 V, Eacc = 0, tacc = 15 s and υ = 50 mV/s.

The Ip increased with increasing pH and the highest peak ( 52 nA at Ep of -1192 mV) was obtained in pH 9.0. The Ip sharply decreased at pH 10.0 to 12.0 and no peak was significantly appeared the pH 13.0. These results show that the reduction of AFB2 was totally depends on the pH of supporting electrolyte. The Ep of AFB2 was shifted toward 5

more negative direction with increasing of the pH of the BRB solution which indicates that in more alkali solution, the reduction of AFB2 was much difficult to take place.

3.2 Optimisation of conditions for the stripping analysis The voltammetric determination of analyte at trace level normally involves very small current response. For that reason it is important to optimise all the parameters which may have influence on the measured current. The effect of initial potential ( Ei), final potential (Ep) accumulation potential (Eacc ), accumulation time (tacc ), scan rate (υ ) and pulse amplitude to the Ep and Ip of the AFB2 peak have been studied in BRB pH 9.0. In this optimisation procedures, 0.06 uM AFB2 was used with initial parameters as follows; Ei = -1000 mV, Ef = -1500 mV, Eacc = -800 mV, tacc = 80 s, υ = 40 mV/s and pulse amplitude = 100 mV/s. Using these parameters, the Ip and Ep of 0.06 uM AFB2 were found to be 13.90 nA and -1250 mV ( vs Ag/AgCl ) respectively. Effect of Eacc, Ei and Ef For Ei = -1000 mV and Ef = -1500 mV, the effect of Eacc on the Ip and Ep of AFB2 was studied. The results revealed that the Ip of AFB2 obtained by using Eacc of -200 mV, -400 mV, -600 mV, -800 mV and -1000 mV were not significantly different which were about 14 nA. To get the maximum response, the Ef was changed from -1500 mV to -1400 mV. The result is shown in Figure 2.0

25 A

Ip (nA)

20

B

15 10 5 0 200

400

600

800

1000

Accum ulation potential (-m V)

Figure 2.0: Effect of Eacc on the Ip of 0.06 uM AFB2 obtained using different scan potential windows. (A) Ei = -1000 mV, Ef = -1500 mV and (B) Ei = -1000 mV, Ef = -1400mV. Using Ef = -1400 mV, the optimum Eacc was -600mV which gave the Ip of 16.84 nA. The Ep of AFB2 was not changed with different Eacc used in this experiment. Effect of tacc

6

The effect of the tacc to the Ip of AFB2 (Figure 3.0) shows that by increasing tacc , the Ip linearly increases following this equation:( R2 = 0.9990, n = 5)

Ip (nA) = 0.4038x ( x s ) - 0.2847

for the range of 20 to 80 s. The maximum peak current ( 31.86 nA) was obtained when tacc = 80 s. When AFB2 was longer deposited at mercury electrode, the Ip was not significantly increased, hence the tacc at 80s was chosen as the optimum tacc. The Ep of AFB2 shifted toward less negative direction with increasing tacc.

50 Ip (nA )

40 30 20 10

[AFB2]=0.6X10-7M

0 0

30

60

90

120

150

Accumulation time (s)

Figure 3.0: Effect of tacc on the Ip of 0.06 uM, Ei = - 1000 mV, Ef = -1400 mV, tacc = 80 s, υ = 40 mV/s and P.A = 100 mV. Effect of υ

1225

50 40 30 20 10 0

E p (-m V )

Ip (nA)

Relationship between υ and the Ip of the AFB2 also was investigated which revealed that when the υ increased, the Ip increased as shown in Figure 4a. The Ip was not significantly different when the υ was more than 60 mV/s. Therefore, 50 mV/s has been selected as the best υ for determination of AFB2 in BRB pH 9.0. The peak potential of AFB2 has linearly shifted toward more negative direction for the range of 20 to 50 mV/s and became level off for the υ of 60 and 80 mV/s as shown in Figure 4b. For the υ of 50 mV/s, the pulse amplitude has to be changed to 80 mV instead of 100 mV otherwise, the Ip cannot be calculated by software. The maximum Ip obtained from this optimisation was 38.18 nA which was about 200% higher than before being optimised. Figure 6.0 illustrates the voltammogramms of 0.06 uM AFB2 obtained under optimum and unoptimum conditions.

[AFB2]=0.6X10-7M

1220 1215 1210 1205

[AFB2]=0.6X10-7M

7

1200

0

20

40

60

Scan rate (mV/s)

80

100

0

20

40

60

Scan rate (mV/s)

80

100

(a)

(b)

Figure 4.0: Effect of υ on the Ip (a) and Ep (b) of AFB2. Ei = - 1000 mV, Ef = -1400 mV, Eacc = -600 mV, tacc = 80s and P.A = 100 mV

Figure 5.0: Voltammogramms of 0.06 uM AFB2 obtained under unoptimum (a) and optimum (b) parameters. From this optimisation procedures, the selected optimum parameters for determination of AFB2 compound in BRb pH 9.0 were Ei = -1000 mV, Ef = -1400 mV, Eacc = -600 mV, tacc = 80 s, υ = 50 mV/s, pulse amplitude = 80 mV and quite time = 10 sec.

3.2.4 Calibration curve of AFB2 Using the optimum parameters, a calibration curve has been prepared by a series of standard addition of AFB2 as shown in Figure 6.0. The range of linearity was found to be from 0.02 u M to 0.32 u M with the limit of detection of 0.0158 uM. Figure 7.0 shows the voltammograms of AFB2 obtained by this standard addition.

8

ip (nA)

200 180 160 140 120 100 80 60 40 20 0

y = 5.55x + 3.6435 R2 = 0.9978

0

10

20

30

40

[AFB2] X10-8M

Figure 6.0: Linear plot of Ip versus concentration of AFB2 in BRb pH=9.0

Figure 7.0:

Cathodic stripping voltammograms for increasing concentration of AFB2 (1) background (2) 0.02 (3) 0.06 (4) 0.10 (5) 0.14 (6) 0.18 (7) 0.22 (8) 0.26 and (9) 0.32 uM. E acc = -600 mV, t acc = 80 s, υ = 50 mV/s, P.A = 80 mV and BRB of pH 9.0

For determination of accuracy of the proposed method, the reproducibility was calculated from 8 independent runs of 0.06 uM and 0.20 uM of AFB2 solution, obtaining the relative standard deviation (RSD) of 2.80 % and 2.53 % respectively. In order to check the precision of the method, a recovery study has also been carried out by adding of the different concentration of AFB2 (0.10 uM and 0.25 uM) into the voltammetric cell and measuring the peak currents of the respective concentrations. The actual amount of AFB2 found in the cell were calculated using a regression equation obtained from prepared calibration curve. The recovery obtained were 86% to 95% with RSD of 1.68% and 0.86% ( n = 3) respectively as shown in Table 3.1. The recovery study using real sample will be performed in future work..

9

Table 1.0: Mean values for the recovery of AFB2 (n =3) in prepared AFB2 standard solution ( n = 3 ) No of experiment

Amount added

Peak current

Amount found

Recovery (%)

( uM)

(nA)

(uM)

1

0.10

51.85

8.683

86.83 +/- 1.46

2

0.25

134.43

23.56

94.26+/0.81

4. CONCLUSION The use of the DPCSV technique has been proved to be a useful, simple, accurate and efficient alternative in the quantitative assay of trace amounts of AFB2. The sensitivity of this approach is much greater than obtained by differential pulse polarographic technique (12). The optimised parameters will be applied to determine other aflatoxin compounds such as AFB1, AFG1 and AFG2. Further experiment will be performed to increase it’s sensitivity in future work together with a recovery study of AFB2 using real samples such as ground nut and nut products. An alternative approach will also be studied by using different working electrode such as screen print electrode.

5. ACKNOWLEDGEMENTS The author gratefully acknowledge the Universiti Sains Malaysia (USM) for granting a study leave together with a scholarship under the Academic Staff For Higher Education Scheme (ASHES). Also we would like to thank all Chemistry Department staff, UTM for their fully cooperation. 6. REFERENCES 1. Eaton D.L and Groopman J.D. (Eds) (1994), The toxicity of aflatoxin. Academic Press, United Kingdom. 2.

Goldblatt L.A (1972) Aflatoxin: Scientific background, control and implication, Academic Press, New York (1972)

3. Andeou V.G., Nikolelis D. and Tarus B. (1997). Electrochemistry investigation of transduction of interactions of alfatoixin M1 with bilayer lipid membranes (BLMs), Anal. Chim. Acta, 350. 121-127.

10

4. Jaimez J., Fente C.A., Vazquez B.I., Franco C.M., Cepeda A., Mahuzier G. and Prognon P (2000). Application of the assay of aflatoxins by liquid chromatography with fluorescence detection in food analysis. J. Chromatography A.” 882: 1-10 5. Gimeno A. and Martins M. L (1983) Rapid thin layer chromatographic determination of patulin, citrinin and aflatoxin in apples and pears and their juices and jams, J. Assoc. Anal. Chem, 66, 85-91. 6. Vahl M. and Jorgensen (1998). Determination of aflatoxins in food using LC/MS/MS, Z Lebensm Unters Forsch A , 206. 243-245 7. Kussak A., Anderson B and Anderson K. (1995). Determination of aflatoxins in airborne dust from feed factories by automated immunoaffinity column clean-up and liquid chromatography, J Of Chromatography A, 708. 55-60 8. Pesavento M., Domagala S., Baldini E. and Cucca L. (1997). Characterisation of enzyme linked immunosorbent assay for aflatoxin B1 based on commercial reagents, Talanta, 45. 91-104. 9. Braitina Kh.Z Malakhova N.A. and Stojko Y. (2000) Stripping voltammetry in environmental and food analysis, Fresenius J Anal Chem 368: 307-325 10 Fifield F.W. and D. Kealey (2000), Principle and practice of analytical chemistry 3rd Edition, Blackwell science, UK 11.Yaacob M.H, Yusoff A.R, Ahamad R and Yaakob A.R (2003), Cyclic voltammetric study of aflatoxin B2 at the mercury electrode, Paper presented at 16th Symposium Of Chemical Analysis, 9-11th September 2003, Kucing, Sarawak, Malaysia. 12. Smyth M. R., Lawellin D. W. and Osteryoung J.G (1979) Polarographic Study of Aflatoxin B1, B2, G1 and G2: Application of Differential-pulse Polarography to the Determination of Aflatoxin B1 in Various Foodstuffs, Analyst, 104: 73-78

11

Paper will be published in

Malaysian Journal of Analytical Science

CYCLIC VOLTAMMETRIC STUDY OF AFLATOXIN B2 AT THE MERCURY ELECTRODE Mohamad Hadzri Yaacoba, Abdull Rahim Hj. Mohd. Yusoffb and Rahmalan Ahamadb a b

School Of Health Sciences, USM, 16150 Kubang Krian, Kelantan Chemistry Dept., Faculty Of Sciences, UTM, 81310 Skudai, Johor

ABSTRACT A cyclic voltammetry (CV) study of aflatoxin B2 (AFB2) in Britton-Robinson buffer using a HMDE is described. CV was carried out by anodic and cathodic potential scan through within the range of 0 to -1500 mV with no accumulation time. The effect of the different scan rate on the peak height and peak potential of the analyte were also studied. The results from this study showed that the reduction process on the hanging mercury electrode gives a single characteristic cathodic peak at -1315 mV ( versus Ag/AgCl ) in pH of 9.0. Effect of the scan rate on both responses has proved that the reduction of AFB2 is irreversible reaction and the limiting current is adsorption controlled. Keywords: aflatoxin B2 compound, cyclic voltammetry, Britton-Robinson buffer and hanging mercury drop electrode (HMDE)

1. INTRODUCTION Aflatoxins are mycotoxins produced by certain fungi, especially, Aspergillus flavus and Aspergillus parasiticus which are found on crops and foodstuffs under certain conditions (1). They were first identified as the causative agent of the severe outbreak of “Turkey X” disease, a toxicosis that killed more than 100,000 turkey poults in England in 1960 (2) They display strong carcinogenicity. Therefore, they are dangerous food contaminants and many countries including Malaysia have set regulatory demands on the level of aflatoxins permitted in imported and trade commodities. Aflatoxin B2 (AFB2) is one of the naturally occurrence aflatoxins. The chemical structure of this compound is shown in Figure 1.0.

a

Corresponding Address: Chemistry Dept, Faculty of Science, UTM, 81300 Skudai, Johor Tel: 07- 553 4540 e-mail: [email protected]

2

O O

O

O

O O

Figure 1.0: Chemical structure of AFB2 Due to its toxicity, it is important that the level of this contaminant be controlled in food, soils and water to prevent toxic effect. The way to achieve this is to monitor samples so that any problem can be identified and dealt with by doing analysis of aflatoxin using the analytical techniques that are necessary to determine aflatoxin at the parts-per-billon (ppb) level. Currently, analysis is done by various methods including thin-layer chromatography (TLC) (3), liquid chromatography (LC) and high-performance liquid chromatography (4,5) and microtitre plate enzyme-linked immunosorbant assay (ELISA) (6). All these methods are time consuming and require special equipments operated by skilled personnel. The proposed voltammetric technique generally refer to a system that combines a triple electrodes system with potentiostat for controlling applied potential. This technique is rapid, simple and relative low cost (7). It measures the current response generated by mass transport of electroactive species in solution to the working electrode while potential is scanned The total current response is proportional to the amount of analyte present in the solution (8). This paper reports the results from the cyclic voltammetric study of AFB2 at a mercury electrode.

2. EXPERIMENT 2.1

Reagents

All reagents employed were of analytical grade. Water purified from a Nano Pure Ultrapure water system ( Barnstead / Thermolyne) was used for all dilution and sample preparation. AFB2 was purchased from SIGMA: 1 mg was dissolved in hexane: acetonitrile (98:2) to produce 10 ppm each. The stock solutions were placed in small glass tubes covered with an aluminum foil and kept in a freezer at -4.0 oC. A stock of Britton-Robinson Buffer (BRB) solution 0.04 M was prepared as follows: 2.47 g boric acid (Fluka), 2.30 ml acetic acid (MERCK) and 2.70 ml orthoposphoric acid ( Ashland Chemical ) and were diluted to 1 l with deionised water. Hexadistilled mercury, grade

3

9N, used by the Controlled Growth Mercury Electrode (CGME) stand, was purchased from MERCK. The purging was carried out with nitrogen gas with purity 99.99%.

2.2

Instruments

A BAS CV-50W voltammetric analyser in connection with a Control Growth Mercury Electrode (CGME) stand equipped with a three-electrode system and a 20 ml capacity BAS MR-1208 cell was used for all the voltammetric determination. The working electrode was a Hanging Mercury Drop Electrode (HMDE). As reference and counter-electrode, a silver-silver chloride (Ag / AgCl) and a platinum wire were used, respectively. All potentials are quoted relative to this reference electrode. BAS CV-50W was connected to a computer for data processing. A pH meter Cyber-scan model equipped with a glass electrode combined with an Ag/AgCl reference electrode, was employed for the pH measurements.

2.3

Procedure

Cyclic voltammetry was studied within the range of 0 to -1500 mV with different scan rate from 20 to 500 mV/s in BRB pH 9.0 as the supporting electrolyte. The HMDE and Ag/AgCl were used as the working and reference electrode respectively through out this experiment . The general procedure used to obtain cyclic voltammograms was as follows. A 10 ml aliquot of buffer solution was placed in a voltammographic cell using a micropipette. The stir was switched on and the solution was purged with nitrogen gas for 10 minute. After forming a new HMDE, potential scan was effected using different scan rate while stirring the solution. For the aflatoxin standard stock solution, organic solvent was removed by flowing nitrogen gas to dryness followed by adding of the BRB supporting electrolyte solution. When further volume of aflatoxin were added to the cell, the solution was redeoxygenated with nitrogen gas for 5 minute before carried out further voltammetry followed the mentioned procedures.

3. RESULTS AND DISCUSSION Cathodic cyclic voltammogrammes of 1.3 X 10-6 M AFB2 in BRB solution at pH=9.0 and a scan rate (υ) of 200 mV/s can be observed in Figure 2.0. A sharp and well defined cathodic wave around -1.315 V ( against Ag/AgCl ) with a peak current of 30 nA is observed due to the reduction of the AFB2. No oxidation wave appears in the anodic branch, which indicates that the AFB2 reduction is irreversible. This was confirmed by anodic cyclic voltammetric that gave the same results as shown in Figure 3.0

4

Figure 2.0: Cathodic cyclic voltammogram for 1.3 X10-6 M AFB2, obtained at υ of 200 mV/s, Ei = 0, Elow = -1.5 V and Ehigh = 0 in BRB solution at pH=9.0. ( 1 ) cathodic and (2) anodic scans

Figure 3.0: Anodic cyclic voltammogram for 1.3 X10-6 M AFB2, obtained at υ of 200 mV/s, Ei = -1.5, Elow = -1.5 V and Ehigh = 0 in BRB solution at pH=9.0. ( 1 ) anodic and (2) cathodic scans

Standard additions of AFB2 was carried out to confirm that the observed peak is related to the reduction of AFB2. Figure 4.0 shows that the Ep at around -1.315 V is referred to the AFB2 peak since there are no significant change in location of the peak and the Ip increases with increasing of the AFB2 concentration. There are no extra peak apparently observed with this standard addition. The corresponding equation of this

5

dependence of Ip to concentration of AFB2 for cathodic cyclic voltammetric is at pH =9.0, ( r = 0.9817, n = 4 ) Ip (nA) = 23.839 x ( x 10 -6 M) + 4.44 The regression equation shows that the Ip of AFB2 does not very linearly with the concentration of AFB2 suggesting formation of a compact film on the electrode surface (9).

Figure 4.0 Effect of AFB2 concentration on the Ip of cathodic cyclic voltammetric curve in BRB at pH =9.0. (a) 1.3 X10-6 M , (b) 2.0 X 10-6 M (c) 2.7 X10-6 M and (d) 3.4 X 10-6 M. A result from repetitive cathodic stripping voltammetry experiment is shown in Figure 5.0. The result showed that the reduction peak of AFB2 increases with number of cycles indicating irreversible reduction with adsorption of AFB2 at the mercury electrode surface. No other cathodic peak was observed.

6

Figure 5.0: Repetitive cathodic cyclic voltammograms of 1.3 X 10-6 M AFB2 in BRB solution at pH of 9.0. The adsorption of AFB2 on the electrode surface is expected due to the AFB2 chemical structure, that consists of functional groups such as ketone, ester and ether. The presence of these groups increases the polarity and enhance absorption of molecules at the electrode surface (10). Ip increases with the number of cycle. However, a further increases in the repetitive cycle tend to slow down the increment of the Ip due to the deformation of double layer at the mercury electrode surface (11). The effect of increasing scan rate from 20 to 500 mV/s to the Ep and Ip of AFB2 cathodic peak were observed under the same experiment conditions. Linear relationship was observed between the log Ip versus log υ with a slope of 0.5097 ( R2 = 0.995, n = 9 ) as shown in Figure 6.0. The slope of more than 0.5 indicates that the diffusion current is influenced by an adsorption on the electrochemical process at the electrode surface (12, 13). The plot of Ep versus log scan rate which is a linear graph as shown in Figure 7.0 ( R2 = 0.9936, n = 9 ) confirmed that the reduction of AFB2 on the electrode surface is totally irreversible. This irreversible reaction is also confirmed by observing the shifted of Ep towards more negative direction when the υ is increased, according to the following equation: Ep (-mV) = 40.065log υ + 1223.2

( R = 0.9936, n = 9)

1.9 1.7

log Ep

1.5 1.3 1.1 0.9

y = 0.5097x + 0.4115 R2 = 0.995

0.7 0.5 1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

log V

Figure 6.0: Plot of log Ip versus log υ for 1.3 X10-6 M AFB2 in BRB solution at pH 9.0

7

1340

Ep (-mV)

1330 1320 1310 1300 1290

y = 40.065x + 1223.3 R2 = 0.9936

1280 1270 1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

log V

Figure 7.0: Plot of Ep versus log υ for 1.3 X10-6 M AFB2 in BRB solution at pH 9.0

4. CONCLUSION From the CV study the AFB2 reduced at the mercury electrode produced a single and well characteristic cathodic peak at -1.315 V ( versus Ag/AgCl) . The reduction is totally irreversible reaction and the diffusion current is controlled by adsorption process. Further work on stripping voltammetric study of this compound will be carried out.

5. ACKNOWLEDGEMENTS The author gratefully acknowledge the University Science Malaysia for giving me a study leave together with a scholarship under the Academic Staff For Higher Education Scheme (ASHES). Also we would like to thank all Chemistry Department staff, UTM for their fully cooperation.

6. REFERENCES 1. Eaton D.L and Groopman J.D. (Eds) (1994), The toxicity of aflatoxin. Academic Press, United Kingdom. 2. Goldblatt L.A (1972) Aflatoxin: Scientific background, control and implication, Academic Press, New York (1972)

8

3. Gimeno A. and Martins M. L (1983) Rapid thin layer chromatographic determination of patulin, citrinin and aflatoxin in apples and pears and their juices and jams, J. Assoc. Anal. Chem, 66, 85-91. 4. Vahl M. and Jorgensen (1998). Determination of aflatoxins in food using LC/MS/MS, Z Lebensm Unters Forsch A , 206. 243-245 5. Kussak A., Anderson B and Anderson K. (1995). Determination of aflatoxins in airborne dust from feed factories by automated immunoaffinity column clean-up and liquid chromatography, J Chromatography A, 708. 55-60 6. Pesavento M., Domagala S., Baldini E. and Cucca L. (1997). Characterisation of enzyme linked immunosorbent assay for aflatoxin B1 based on commercial reagents, Talanta, 45. 91-104. 7. Braitina Kh.Z Malakhova N.A. and Stojko Y. (2000) Stripping voltammetry in environmental and food analysis, Fresenius J Anal Chem, 368. 307-325 8. Harvey, D (2000). Modern Analytical Chemistry, McGraw Hill, USA 9. Laviron E in Bard A (Eds) (1982) Electroanalytical Chemistry, Marcell Decker, New York . 10. Volke J and Liska F (1994) Electrochemistry In Organic Synthesis, SpringerVerlag, New York. 11. Fifield F.W. and Kealey D (2000) Principle and Practice of Analytical Chemistry 3rd Edition, Balckwell Science, U.K 12. Gosser D.K (1993) Cyclic voltammetry, Wiley-VCH, New York 13. Yaridimer C. and Ozaltin N. (2001) Electrochemical studies and differential pulse polarographic analysis of lansoprazole in pharmaceuticals, Analyst, 126. 361-366

9

PAPER WAS PUBLISHED IN

J. Kimia Analisis (Universiti Sumatera Utara, Medan, Indonesia)

Yaacob, M.H., Mohd. Yusoff, A.R. and Ahamad, R. (2005). Application of Differential Pulse Cathodic Stripping voltammetry (DPCSV) Technique in Studying Stability of Aflatoxins. J. Sains Kimia. 9(3): 64-70.

1

APPLICATION OF DIFFERENTIAL PULSE CATHODIC STRIPPING VOLTAMMETRIC TECHNIQUE IN STUDYING STABILITY OF AFLATOXINS Mohamad Hadzri Yaacob1, Abdull Rahim Hj. Mohd. Yusoff2 and Rahmalan Ahamad2 1

School Of Health Sciences, USM, 16150 Kubang Krian, Kelantan, Malaysia Dept of Chemistry , Faculty of Sciences, UTM, 81310 UTM, Skudai, Johor, Malaysia

2

Abstract A stability studies of aflatoxins (AFB1, AFB2, AFG1 and AFG2 ) in Britton-Robinson buffer (BRb) using a Differential Pulse Cathodic Stripping Voltammetric (DPCSV) technique is described. The DPCSV was performed by cathodic potential scan through within the range of -950 to -1400 mV with 80s accumulation time using a BRb at pH 9.0 as the supporting electrolyte. The samples were exposed for 0 to 8 hrs to normal laboratory condition before being scanned under optimised voltammetric parameters. Using the same procedure, aflatoxins in different pH of BRb were also studied after 0 to 3 hours exposed to the same condition. Other stability study was performed by scanning aflatoxins in BRb solution which were kept in freezer from first to six months of storage time. The results from these studies showed that AFB1 and AFB2 in BRb pH 9.0 were stable within 8 hours exposure time while AFG1 and AFG2 were stable up to a few hours. All aflatoxins scanned in BRB pH 6.0, 7.0 and 9.0 were more stable as compared when there were scanned in BRB pH 11.0 within 3 hours exposure time. The results also showed that the stability of the samples which were prepared in BRB pH 9.0 was affected by longer storage time in freezer.

Abstrak Satu kajian terhadap kestabilan aflatoksin (AFB1, AFB2, AFG1 and AFG2 ) di dalam larutan penimbal Britton-Robinson (BRb) menggunakan teknik Voltammetri Perlucutan Kethodik Denyut Pembeza (DPCSV) adalah dilaporkan. Kaedah ini dijalankan dengan cara mengimbas keupayaan katodik di dalam julat di antara -950 ke -1400 mV dengan masa penjerapan 80 s menggunakan larutan penimbal pada pH 9.0 sebagai larutan penyokong. Sampel didedahkan kepada persekitaran makmal dari 0 hingga 8 jam sebelum diimbas menggunakan parameter voltammetri yang optimum. Menggunakan prosedur yang sama, aflatoksin yang disediakan di dalam larutan penimbal yang berbeza pH juga dikaji kestabilan dari 0 hingga 3 jam terdedah kepada kondisi yang sama. Kajian kestabilan yang lain yang dijalankan termasuk mengimbas aflatoksin yang disediakan di dalam larutan penimbal dan disimpan di dalam penyejukbeku dari bulan pertama hingga ke bulan keenam. Keputusan dari kajian ini menunjukkan bahawa AFB1 dan AFB2 di dalam larutan BRb stabil di dalam tempuh 8 jam terdedah kepada keadaan biasa makmal dan AFG1 serta AFG2 hanya stabil di dalam tempuh beberapa jam sahaja. Semua aflatoksin yang diimbas di dalam pH 6.0, 7.0 dan 9.0 di dalam tempuh 3 jam masa pendedahan adalah lebih stabil berbanding jika ianya diimbas di dalam pH 11.0. Keputusan juga menunjukkan bahawa kestabilan sampel yang disediakan di dalam BRb dengan pH 9.0 adalah dipengaruhi oleh jangkamasa simpanan di dalam penyejukbeku.

Keywords: AFB1, AFB2, AFG1, AFG2 compounds, stability studies and differential pulse cathodic stripping voltammetry

INTRODUCTION Aflatoxins are a group of secondary metabolites produced by Aspergillus flavus and Aspergillus parisiticus, which are found on crops and foodstuffs under certain conditions [[1]. Aflatoxins B1, B2, G1 and G2 have been found to be naturally and they display strong carcinogenicity. There are dangerous food contaminants and have been classified as Type I toxic materials by International Agency for Research 1

Corresponding Address: Chemistry Dept, Faculty of Science, UTM, 81300 Skudai, Johor Tel: 07- 553 4540 or 019-939 2440 or e-mail: [email protected]

( Note: A part of this paper was presented in Simposium Kimia Analisis Malaysia ke 17 (SKAM-17), 24 – 26th August 2004 at Kuantan, Pahang, Malaysia)

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Cancer (IARC) in 1984 [2]. Due to its toxicity, many countries have set stringent regulatory demands on the level of aflatoxins permitted and traded commodities. In our country, the regulatory levels for total aflatoxins in raw peanuts are 15 ppb and 5 ppb for other foodstuffs [3].Aflatoxins are not totally diminished by normal and cooking temperatures [4,5]. The chemical structures of AFB1, AFB2, AFG1 and AFG2 are shown in Figure 1.0 [6].

O O

O O

O O

O

O

O

O

O

O

(a)

(b)

O

O

O

O

O

O

O

O

O

O

O

O

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O

O

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Figure 1.0: Chemical structures of (a) AFB1, (b) AFB2, (c) AFG1 and (d) AFG2

Many methods have been used for quantitative determination of aflatoxins includes thin layer chromatography (TLC) [7-9], high performance liquid chromatography (HPLC) with different type of detectors such as ultra-violet [10-12], fluorescence [13-15] and amperometer detectors [ 16]

In this study, all aflatoxins have been investigated for its stability in BRB pH 9.0 using DPSV technique. This technique was chosen rather than UV-VIS spectrophotometer because it uses the principle of the measurement of peak height of electro active species in sample solution when potential was applied into it [17]. It can avoid problem arise from weak fluorescence property of AFB1 and AFG1 [16]. This technique also offers shorter time analysis compare to the HPLC technique [17].

In the present paper, we describe the results of stability studies on AFB1, AFB2, AFG1 and AFG2 which were prepared in BRB pH 9.0 and underwent hour to hour monitoring and also for shelf life of all aflatoxins in BRB pH 9.0 which was kept in fridge for six months.

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EXPERIMENTAL Reagents All reagents employed were of analytical grade. Water purified from a Nano Pure Ultrapure water system ( Barnstead / Thermolyne) was used for all dilution and sample preparation. Aflatoxins were purchased from SIGMA ( 1 mg per bottle ) and dissolved in benzene:acetonitrile (98:2) to produce 10 ppm standard solution each. The stock solutions were placed in small amber glass tubes and kept in a freezer at 4.0oC. The solution was measured under UV-VIS spectrometer for it absorbance at 365 +/- nm before being used for voltammetric analysis. A stock solution of 0.04 M Britton-Robinson Buffer (BRB) solution was prepared as follows: 2.47 g boric acid (Fluka), 2.30 ml acetic acid (MERCK) and 2.70 ml orthophosphoric acid ( Ashland Chemical ) were diluted to 1 l with deionised water. Britton-Robinson Buffer of pH 5, 7.0 and 11.0 were prepared by adding sodium hydroxide 1.0 M (MERCK) into the stock solution.. Hexadistilled mercury, grade 9 N, used in a control growth mercury electrode ( CGME ) stand, was purchased from MERCK. The purging was carried out with 99.99% nitrogen. Instruments A BAS CV-50W voltammetric analyser in connection with a CGME stand equipped with a threeelectrode system and a 20 ml capacity BAS MR-1208 cell were used for all the voltammetric determination. The working electrode was a hanging mercury drop electrode (HMDE). A silver-silver chloride (Ag / AgCl) and a platinum wire were used as the reference and counter-electrode respectively. BAS CV-50W was connected to a computer for data processing. A pH meter Cyber-scan model equipped with a glass electrode combined with an Ag/AgCl reference electrode, was used for all pH measurements. Procedure General procedure The general procedure used to obtain cathodic stripping voltammograms was as follows. A 10 ml aliquot of supporting electrolyte solution was put in a voltammographic cell. The stir was switched on and the solution was purged with nitrogen gas for at least 10 minutes. After forming a new HMDE, accumulate was effected for the required time at the appropriate potential while stirring the solution. At the end of the accumulation time the stirring was switched off and after 10 s had elapsed to allow the solution to become quiescent, the negative going potential was initiated. Ag/AgCl saturated was used as the reference electrode throughout this study.

Procedure for determination of aflatoxin The aflatoxin standard solutions have been freshly prepared by removing of organic solvent in aflatoxin stock solutions until dryness followed by the addition of the BRb supporting electrolyte solution. The solution was placed in small amber bottles. When further volume of aflatoxin were added using a micropipette into the voltammetric cell, the solution was purged with nitrogen for another 2 minutes before further voltammetric experiment was carried out. Procedure for stability studies For hour to hour stability studies, 312 ul, 315 ul, 328 ul and 330 ul of 1 ppm AFB1, AFB2, AFG1 and AFG2 standard solution in BRB pH 9.0 respectively were injected into 10 ml supporting electrolyte in the voltammetric cell separately and were scanned following the above stated procedures.

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Eight measurements have been made. The peak heights and peak potential of each aflatoxins were recorded. After these measurements, the aflatoxins standard solutions in voltammetric cell were exposed to normal laboratory condition without purging. Measurements were taken every hour up to 8 hours exposure time. For shelf life stability studies, all aflatoxin standard solutions which were kept in freezer were subjected to the voltammetric measurement in BRb pH 9.0 every month up to 6 months keeping period.

For aflatoxins in different pH of BRB, the stability studies were performed by scanning aflatoxins solution (prepared in BRB pH 9.0) in four difference pH of BRB as the supporting electrolytes which were 6.0, 7.0, 9.0 and 11.0. The voltammetric determination procedures were followed the same procedures as for the hour to hour stability studies except the exposure time was limited up to 3 hours.

RESULTS AND DISCUSSION From our previous experiment using a cyclic voltammetry technique , AFB1, AFB2, AFG1 and AFG2 show an irreversible reduction peak at -1235 +/- 10 mV, ( all against Ag/AgCl) respectively [18, 19]. Based on this electro active property, differential pulse cathodic stripping voltammetry (DPCSV) technique was applied in this study. Voltammograms of aflatoxins The voltamograms of 0.1 uM AFB1, AFB2, AFG1 and AFG2 scanned in BRb pH = 9.0 are shown in figures 2.0a -2.0d. Replicate measurements of peak height of respective aflatoxins give the repeatability results of 63.19 nA, 65.67 nA, 60.76 nA and 61.79 nA respectively with coefficient variation of 1.50 to 2.60 % for all aflatoxins. The results show that the DPSV technique give high consistent results with very small error and could be applied in stability studies of aflatoxins.

(a)

(c)

(b)

(d)

Figure 2.0 :Voltammograms ( n = 8) of 0.1 uM AFB1 (a), AFB2 (b), AFG1 (c) and AFG2 (d) in BRb pH = 9.0.

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Hour to hour stability studies; The results of these studies using 0.10 uM AFB1, AFB2, AFG1 and AFG2 in BRB pH 9.0 are represented in Figure 3.0. 70 Peak height (nA)

60 50

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10 0 0

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Figure 3.0: Peak heights of AFB1, AFB2, AFG1 and AFG2 in BRb pH = 9.0 exposed to normal condition up to 8 hours.

The results show that AFB1 and AFB2 in BRb pH 9.0 were stable within 8 hours exposure time where the peak heights for both aflatoxins were reduced only 15%. In contrast, AFG1 and AFG2 gave significant reduced of peak heights within the same period of exposure time where the peak height of AFG1 and AFG2 were reduced to 50% and 58% respectively. The results show that AFG1 and AFG1 were less stable in BRb pH 9.0 as compared to AFB1 and AFB2. Based on their chemical structures, the most reactive functional groups for ease of attack by chemical reagents are two lactones rings of AFB1 and AFG1 and cyclopentanone rings in AFB2 and AFG2. These lactones can be readily opened by hydrolysis with strong alkalis such as sodium hydroxide. Other functional group of this aflatoxin is less readily attacked by chemical reagents. The methyl ether and furan ether groups would be cleaved only by very strong acids such as hydriodic acid. The double bond of terminal furan rings of AFB1 and AFG1 are susceptible to attack by electrophilic reagents and can be reduced or oxidised [20]. Shelf life stability studies For shelf life stability studies, the results are shown in Figure 4.0 80

Peak height (nA)

70 60 AFB1 50

AFB2 AFG1

40

AFG2 30 20 10 0

1

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4

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Figure 4.0: Peak heights of aflatoxins in BRb pH 9.0 which were kept up to 6 months in freezer.

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The results showed that after three months storage in freezer, the peak heights of AFG1 and AFG2 in BRB pH 9.0 were decreased to about 70% from original peak height measured at first day of preparation. AFB2 was the most stable within 2 months keeping period. These results revealed that AFB1 and AFB2 were stable within 3 months kept in fridge but gradually decreased after that time. Peak heights of AFG1 and AFG1 were gradually decreased since first month keeping time up to 6 months. Stability studies in different buffer solutions The results for these studies are shown in Figure 5.0a to 5.0d. The results show that in acid and neutral conditions, peak heights of all aflatoxins were increased when the exposure time were longer. In BRb pH 9.0, the peak heights were not significantly affected by longer exposure time. This phenomenon may be because of all aflatoxins need enough time to be completely dissolved in acid and neutral condition compared in basic medium. In strong base, the peak height was significantly decreased where AFG2 has not given any peak after 3 hours exposure time. These results indicate that in neutral and acid conditions, aflatoxins take time to stabilise themselves and at strong base, it was slowly reacted while cyclopentanone and lactone groups slowly damaged (21). AFG1 and AFG2 show decreasing in peak height faster than aFB1 and AFB2. This was due to the easy of lactones group in both compounds attacked in strong basic condition compared to acidic condition. Other chemical group was not affected in acid or neutral medium as can be observed from the peak heights of all aflatoxins in BRB pH 6.0 and 7.0 within 3 hours standing time. At 0 hrs standing time, the peak heights of all aflatoxins in BRB pH 6.0, 7.0 and 11.0 were lower as compared in BRb pH 9.0 due to those pH were not an optimum pH for DPCSV analysis of aflatoxins as found in our previous experiment [22].

70

70

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Figure 4.0: Peak heights of 0.1 uM aflatoxins in BRb at pH (a) 6.0, (b) 7.0, (c) 9.0 and (d) 11.0 exposed to normal laboratory condition up to 3 hrs standing time.

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CONCLUSION Using DPCSV technique, stability studies of aflatoxins have been successfully performed. It was concluded that AFB1 and AFB2 in BRb pH 9.0 which were exposed to normal condition were more stable as compared to AFG1 and AFG2 in the same medium. Shelf life of AFB1 and AFB2 keeping in freezer were longer compared to AFG1 and AFG2. All aflatoxins in BRb pH 6.0, 7.0 and 9.0 which were exposed to normal condition up to three hours were stable but in BRb pH 11.0, their peak heights were significantly decreased.

ACKNOWLEDGEMENT One of the author is gratefully acknowledge the University Science Malaysia for approved him a study leave and financial support for his study at UTM. Also we would like to thank UTM for a fundamental research grant (Vot No 75152) to carry out this study.

REFERENCES 1.

Begum F and Samajpathi N (2000), Mycotoxin production in rice, pulses and oilseeds,Naturwissenschaften, 87: 275-277

2.

International Agency for Research on Cancer (1993) IARC monographs on the evaluation of carcinogenic risk to humans, vol. 56. World Health Organisation, Lyon.

3.

Malaysian Food Act (1983) Act No 281, Reviewed 2002, Food Quality Control Dept, Ministry Of Health, Malaysia

4.

Creepy E.E (2002). Update of survey, regulation and toxic effect of mycotoxins Europe, Toxicology Letter, 127: 19-28

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Samarajeewa U., Sen A.C., Cohen M.D. and Wei C.L (1990). Detoxification of Aflatoxins in foods and feeds by physical and chemical methods Food Protection, 53, 489-501.

6.

Goldblatt L.A (1969) Aflatoxin: Scientific background, control and implication, Academic Press, New York

7.

Gimeno A. and Martins M. L (1983) Rapid thin layer chromatographic determination of patulin, citrinin and aflatoxin in apples and pears and their juices and jams, J. Assoc. Anal. Chem, 66, 85-91.

8.

Bicking M.K.L., Kniseley R.N and Svec H.J (1983), Coupled-Column for quantitating low levels of aflatoxins, J . Assoc. Off. Anal. Chem. 66, 905-908.

9.

Tutour L.B., Elaraki A. T. and Aboussalim A.,(1984). Simultaneous thin layer chromatographic determination of aflatoxin B1 and ochratoxin A in black olives, J Assoc Off Anal Chem 67: 611-620.

10. Rastogi S., Das M. and Khanna S.K. (2001). Quantitative determination of aflatoxin B1-oxime by column liquid chromatography with ultraviolet detection. J Of Chromatography A, 933: 91-97 11. Joshua, H (1993) Determination of aflatoxin by reversed-phase high performance liquid chromatography with post-column in-line photochemical derivatization and fluorescence detection, J. Chromatography A, 654: 247-254 12 Miller N, Pretorius H.E. and Trinder D.W (1985). Determination of aflatoxins in vegetable oils, J. Assoc. Off. Anal. Chem, 68 : 136-137 13. Scholten J.M and Spanjer M.C (1996) Determination of aflatoxin B1 in pistachio kernels and shells, , J. Assoc. Off. Anal. Chem 79: 1360-1364

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14. Brera C., Caputi R., Miraglia M., Iavicoli I., Salerno A. and Carelli G. (2002). Exposure assessment to mycotoxins in workplace: aflatoxins and ochratoxin A occurrence in airborne dusts and human sera, Micro chemical J (uncorrected proof). 15. Garcia-Villanova R.J., Cordon C., Paramas A.M.G., Aparicio P. and Rosales M.E.G., (2004). Simultaneous immunoaffinity column clean up and HPLC analysis of aflatoxins and ochratoxin A in Spanish bee pollen, J Agric. Food Chem.52; 7235-7239 16. Gonzalez M.P.E., Mattusch J. and Wennrich R ( 1998) Stability and determination of aflatoxins by highperformance liquid chromatography with amperometric detection, J.Chromatography A, 828: 439-444 17. Fifield F.W. and Kealey D (2000) Principle and practice of analytical chemistry 3rd Edition, Blackwell science, UK 18. Yaacob M.H., Mohd Yusof A.R., and Ahamad R (2003), Cyclic Voltammetry of AFB2 at mercury electrode, Paper presented at SKAM –VI, Unimas, Kucing, Sarawak, Malaysia, 9-11 September 2003 19. Yaacob M.H., Mohd Yusof A.R., and Ahamad R. (2004), Cyclic Voltammetry of AFG1 at mercury electrode, Paper presented at 3rd Annual Seminar on Sustainable Science and Management, KUSTEM, Terengganu, Malaysia, 4 – 5 May 2004. 20. Jaimez J., Fente C.A., Vazquez B.I., Franco C.M., Cepeda A., Mahuzier G. and Prognon P (2000). “Application of the assay of aflatoxins by liquid chromatography with fluorescence detection in food analysis.” J. Chromatography A.” 882: 1-10 21. Miller K (1987) Toxicology aspects of food. Elsevier Applied Science, New York 2.2. Yaacob M.H., Mohd Yusof A.R. and Ahamad R (2005). Differential pulse stripping voltammetric (DPSV) technique for determination of AFB2 at the mercury electrode. To be published in Collection of Czechoslovak Chemical Communication. In process.

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PAPER WAS PUBLISHED IN

J. Kimia Analisis (Universiti Sumatera Utara, Medan, Indonesia)

Yaacob, M.H., Mohd. Yusoff, A.R. and Ahamad, R. (2005). Development of Differential Pulse Cathodic Stripping voltammetry (DPCSV) Technique for the Determination of AFB1 in Ground Nut Samples. J. Sains Kimia. 9(3): 31-36.

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DETERMINATION OF THE AFLATOXIN B1 IN GROUND NUT BY DIFFERENTIAL PULSE CATHODIC STRIPPING VOLTAMMETRY (DPCSV) TECHNIQUE Mohamad Hadzri Yaacob1, Abdull Rahim Hj. Mohd. Yusoff2, Rahmalan Ahamad2 and Marpongahton Mismi3 1

School of Health Sciences, USM, 16150 Kubang Krian, Kelantan, Malaysia Dept. of Chemistry, Faculty of Sciences, UTM, 81310 UTM, Skudai, Johor, Malaysia 3 Dept. of Chemistry, Faculty of Mathematic and Science, USU, Medan, Indonesia 2

ABSTRACT An electro analytical method has been developed for the detection and determination of the 2,3,6a,9a-tetrahydro-4-methoxycyclo penta[c] furo[3’,2’:4,5] furo [2,3-h][l] benzopyran-1,11-dione (aflatoxin B1, AFB1) by a differential pulse cathodic stripping voltammetry on a hanging mercury drop electrode (HMDE) in aqueous solution with Britton-Robinson buffer (BRb) as supporting electrolyte. Effect of instrumental parameters such as accumulation potential (Eacc), accumulation time (tacc) and scan rate (v) were examined. The best condition were found to be pH 9.0, Eacc of -0.6 V, tacc of 80 s and v; 50 mV/s. Calibration curve is linear in the range of 0.02 to 0.32 uM with a detection limit of 0.75 x 10-8 M (3SDblank/m). Relative standard deviation for a replicate measurements of AFB1 ( n= 8) with the concentrations of 0.02 and 0.20 uM were 1.27% and 0.83% respectively. The method is applied for determination of the AFB1 in ground nut samples after extraction and clean-up steps. The recovery values obtained in spiked ground nut sample are 90.93 +/- 2.53 % for 3.0 ppb and 86.15 +/- 2.03 % for 15.0 ppb of AFB1.

ABSTRAK Satu kaedah elektroanalisis telah dibangunkan untuk mengesan dan menentukan 2,3,6a,9a-tetrahydro-4-methoxycyclo penta[c] furo[3’,2’:4,5] furo [2,3-h][l] benzopyran1,11-dione (aflatoxin B1, AFB1) menggunakan teknik voltammetri perlucutan katodik denyut pembeza di atas elektrod titisan raksa tergantung (HMDE) di dalam larutan akuas dengan larutan penimbal Britton-Robinson bertindak sebagai larutan penyokong. Kesan parameter peralatan seperti keupayaan pengumpulan (Eacc), masa pengumpulan (tacc) dan kadar imbasan (v) telah dikaji. Keadaan terbaik yang diperolehi adalah pH 9.0, Eacc; -0.6 V, tacc; 80 s dan v; 50 mV/s. Keluk kalibrasi adalah linear pada julat di antara 0.02 ke 0.32 uM dengan had pengesan pada 0.75 x 10-8 M (3SDblank/ m). Sisihan piawai relatif 1

Corresponding address: Chemistry Dept, Faculty of Science, UTM, 81300 Skudai, Johor, Malaysia tel: 07-553 4492 or 016-926 0354 or e-mail address: [email protected]

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untuk pengukuran AFB1 dengan kepekatan 0.02 dan 0.2 M sebanyak 8 kali ialah 1.27 % dan 0.83 % masing-masingnya. Kaedah ini telah digunakan untuk menentukan kandungan AFB1 di dalam sampel kacang tanah selepas proses pengekstraksian dan pembersihan. Nilai perolehan semula di dalam sampel kacang yang disuntik dengan 3.0 ppb dan 15.0 ppb AFB1 adalah 90.93 +/- 2.53 % dan 86.15 +/- 2.03 % masingmasingnya. Keywords: Cathodic stripping voltammetry; aflatoxin B1; ground nut

INTRODUCTION Aflatoxins are mycotoxin produced by certain fungi especially, Aspergillus flavus and parasiticus , and they display strong carcinogenicity [1]. Therefore, they are dangerous food contaminants and many countries have stringent regulatory demands on the level of aflatoxins permitted in imported and traded commodities. Aflatoxin B1, B2, G1 and G2 are the main toxins and may be present in many foodstuff or animal feed such as maize, peanuts, nuts, copra and cottonseeds [2]. Of these, AFB1 (Fig 1) is the most commonly occurring variety and one of the most toxic [3]. The order of toxicity , AFB1 > AFG1 > AFB2 > AFG2, indicates that the terminal furan moiety of AFB1 is a critical point for determining the degree of biological activity of this group of mycotoxins [4]. O O

O

O

O O

Figure 1. Structural formula of AFB1 One of the foodstuff which is most occurrence of AFB1 is ground nut. In Malaysia, the AFB1 level in peanut is regulated with maximum level that cannot be greater than 15 ppb [5]. Several analytical techniques for quantitative determination of the AFB1 in ground nut have been proposed such as thin layer chromatography [6], high performance liquid chromatography, where a derivatisation is required either using trifluoroacetic acid [7], iodine [8] or bromine [9], and an enzyme-linked immunosorbent assay, ELISA [10], All these methods, however, require specialist equipment operated by skilled personnel and expensive instruments and high maintenance cost [11]. Due to all these reasons, a voltammetric technique which is fast, accurate and requires low cost equipment [12] is proposed. This technique uses the concept where the

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peak height of reduction peak of AFB1 is measured when a certain potential is applied [13]. Previous experiment using cyclic voltammetric technique showed that AFB1 reduced at mercury electrode and the reaction is totally irreversible [14]. This work aimed to study and develop a DPCSV method for determination of AFB1 at trace levels and to determine this aflatoxin in ground nut samples. No such report has been published regarding the determination of AFB1 in ground nut using this technique until now.

EXPERIMENTAL Apparatus Voltammetric measurements were performed using BAS CV-50W voltammetric analyzer with Control Growth Mercury Electrode (CGME) stand. The three-electrode system consisted of a CGME as working electrode, Ag/AgCl2 reference electrode and platinum wire as the auxiliary electrode. A 20 ml capacity measuring cell was used for placing supporting electrolyte and sample analytes. All measurements were carried out at room temperature. All pH measurements were made with Cyberscan pH meter, calibrated with standard buffers (pH 4, 7 and 10) at room temperature. Reagents AFB1 standard. (1mg per bottle) was purchased from Sigma Co. and was used without further purification. Stock solution ( 10 ppm) in hexane:acetonitrile (98:2) were prepared and was stored in the dark at -4 0 C. The diluted solution were prepared daily by using certain volume of stock solution, degassed by nitrogen until dryness and redissolved in Britton-Robinson solution at pH 9.0. Britton-Robinson buffer solution was prepared from a stock solution 0.04 M phosphoric (Merck), boric (Merck) and acetic (Merck) acids. BRb solution with other pH also was prepared by adding sodium hydroxide (Merck) 1.0 M up to pH value required. All solutions were prepared in double distilled dionised water. All chemicals were of analytical grade reagents. General procedure For voltammetric experiments, 10 ml of Britton-Robinson buffer solution was placed in a voltammetric cell, through which a nitrogen stream was passed for 600 s before recording the voltammogram. The selected Eacc = - 600 mV was applied during the tacc = 80 s while the solution was kept under stirring. After the accumulation time had elapsed, stirring was stopped and the selected accumulation potential was kept on mercury drop for a rest time (tr = 10 s), after which a potential scan was performed between -0.950 and -1.500 V by DPCSV.

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Procedure for the determination in ground nut samples

Four batches of ground nut included an artificial contaminated have been analysed for AFB1 content. AFB1was extracted from ground nut samples according to the standard procedure developed by Chemistry Department, Penang Branch, Ministry Of Science, Technology and Innovation (MOSTI), Malaysia [15]. 1ml of the final solution from extraction and clean-up steps in chloroform was pipette into an amber bottle, degassed with nitrogen and re-dissolved in 1ml of Britton-Robinson. 200 ul of this solution was spiked into 10 ml supporting electrolyte in volumetric cell. After that, the general procedure was applied and voltammogram of sample was recorded. A 10 ppb of AFB1 standard solution was then spiked into the sample solution before general procedure for voltammetric analysis The content of AFB1 in ground nut sample was calculated based on standard addition method to minimize any matrix effect.

RESULT AND DISCUSSION

Cyclic voltammetric determination of AFB1 Cyclic voltammogram of 1.3 uM AFB1 in Britton-Robinson buffer solution as shown in Figure 2.0 indicates the non-reversibility of the electrode process. It is also confirmed by the study of the influence of the scan rate in this technique, where a linear relationship between peak potential (Ep ) and log scan rate was found as Ep (-mV) = 61.41x + 1171.6 (R2 = 0.9967).

Figure 3.0 Typical repetitive cyclic voltammogram of 1.3 uM AFB1 in BRb pH 9.0; scan rate = 200 mV/s.

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Effect of pH

Ip (nA)

The influence of pH on the voltammetric behaviour of AFB1 has been studied using DPCSV. AFB1 gave a single reduction signal in the pH range of 5.0 to 12.0 where at pH 9.0, the highest peak height has been obtained as shown in Figure 3.0. This can be assigned to the reduction of the ketone group to give the corresponding alcohol [19].

60 50 40 30 20 10 0 4

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Figure 3.0

Effect of pH of BRb on peak height of 1.0 uM AFB1

Optimisation of condition for the stripping analysis The voltammetric determination of analytes at trace levels normally involves very small current response. For that reason it is important to optimise all those parameters which may have an influence on the measured peak height. The effect of various parameters such as accumulation potential (Eacc ), accumulation time (tacc) and scan rate on the peak height of AFB1 have been studied. The results of these optimization were Eacc; -0.6 V, tacc ; 80 s and scan rate 50 mV/s. Other parameters are initial potential (Ei ) = -0.1 V, final potential (Ef ) = -1.4 V and pulse amplitude = 80 mV. Using these optimized parameters, 0.1 uM of AFB1 has produced a single and sharp peak at Ep of 1.21 V with Ip of 60 +/- 5 nA ( n=5) as shown in Figure 4.0.

Figure 4.0 Voltammogram of 0.1 uM AFB1 in BRb pH 9.0 (n=5). Condition; Eacc = -0.6 V, tacc = 80 s and scan rate = 50 mV/s

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Effect of standard addition of AFB1 and analytical characteristics of the method

Using the optimized conditions that already mentioned, a study was made of the relationship between peak current and concentration. There is a linear relationship in the concentration range 0.02 uM and 0.32 uM with a regression equation of y = 5.4363x + 3.7245 ( R2 = 0.9989) as shown in Figure 5.0a and 5.0b. The relative standard deviations (RSD) of the analytical signals at several concentration values were 1.27% and 0.83% for AFB1 at concentration of 0.02 uM and 0.20 uM respectively. The analytical sensitivity has been calculated as 3SDblank / m (16) and was found as 0.75 x 10-8 M. SDblank is the standard deviation from 8 measurements of blank and m is slope from calibration curve.

200

ip (n A )

150 100

y = 5.4363x + 3.7245

50

2

R = 0.9989

0 0

10

20

30

40

[AFB1] x 10-8M

(i)

(ii)

Figure 5.0 (i) Voltammograms of AFB1 with increasing concentration (b) 0.02 uM , (c) 0.04 uM, (d) 0.06 uM, (e) 0.10 uM, (f) 0.14 uM, (g) 0.18 uM (h) 0.22 uM, (i) 0.26 uM, (j) 0.30 uM and (k) 0.32 uM in (a) BRb pH 9.0; (ii) it’s calibration curve. Conditions: Eacc= -0.6 V, tacc = 80 s, scan rate = 50 mV/s.

Determination of AFB1 in ground nut samples

The proposed method was applied to the analysis of the AFB1 in eluate of ground nut samples, spiked with different concentration of AFB1 for recovery studies. In this

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study, the concentrations used were 3.0 ppb and 15.0 ppb The results of this studies are represented in Table 1.0. The results show that the accuracy of the method is good.

Table 1.0 Recovery study of AFB1 spiked in eluate of ground nut samples (n = 3)

No

Amount added (ppb)

Amount found (ppb)

% of recovery

1

3.00

2.73 +/- 0.08

90.93 +/- 2.53

2

15.00

12.92 +/- 0.30

88.15 +/- 2.03

Analyses of AFB1 content in ground nut samples were determined by standard addition in order to eliminate the matrix effects. Voltammograms of ground nut samples are shown in Figures 6(i) to 6(iv). AFB1 was not detected in three samples while in artificial contaminated sample, 333 ppb of AFB1 was found. The results were compared to that obtained by HPLC technique as stated in Table 2.0 which show a good agreement between them. It was concluded that the proposed technique is good, accurate and reliable for determination of AFB1 in ground nut samples.

Table 2.0 The results of DPCSV analysis of AFB1 in ground nut samples compared with HPLC technique.

Sample

By DPCSV technique

By HPLC technique

1 2 3 4

ND ND ND 333 ppb

ND ND ND 337 ppb

Note: ND = not detected

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(i)

(ii)

(iii)

(iv)

Figure 6.0 Voltammograms of ground nut sample No. 1 (i), sample No. 2 (ii), sample No. 3 (iii) and sample No. 4 with standard addition of 10 ppb AFB1 (iv) in BRb pH 9.0 as the blank. Condition; Eacc = -0.6 V, tacc = 80 s and scan rate = 50 mV/s.

ACKNOWLEDGEMENT One of the author would like to thank University Science Malaysia for approving his study leave and financial support to carry out his further study at Dept of Chemistry, UTM. We also indebt to UTM for fundamental short term grant (Vot No 75152) and to Chemistry Department, Penang Branch, Ministry of Science, Technology and Innovation(MOSTI), Malaysia for the procedure of extraction and clean-up of ground nut samples.

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REFERENCES

1. Akiyama H. Goda Y. Tanaka T and Toyoda M. (2001). Determination of aflatoxinsB1, B2, G1 and G2 in spices using a multifunctional column clean-up, J. Of Chromatography A, 932. 153-157. 2. Chiavaro E., Dall’Asta C., Galaverna G., Biancardi A., Gambarelli E., Dossena A. and Marchelli R. (2001). New reversed-phase liquid chromatographic method to detect aflatoxins in food and feed with cyclodextrins as fluorescence enhancers added to the eluent, J Of Chromatography A, 937. 31-40. 3. World Health Organisation in: IARC Monograph on the evaluation of carcinogenic risk to human, WHO, Lyon, 1987. 4. Hall A.J., Wild C.P in: Eaton D.L., Groopman J.D. (Eds). The toxicology of aflatoxins: Human Health, Veterinary and Agricultural Significance, Academic New York, 1994. 5.

Malaysian Food Act (1983) Act No 281, Reviewed 2002, Food Quality Control Dept, Ministry of Health, Malaysia.

6. Bicking M.K.L., Kniseley R.N and Svec H.J (1983), Coupled-Column for quantization low levels of aflatoxins, J . Assoc. Off. Anal. Chem. 66, 905-908. 7. Beebe B.M. (1978). Reverse phase high pressure liquid chromatographic determination of aflatoxins in foods, J. Assoc. Off. Anal. Chem. 81. 1347-1352. 8. Cepeda A., Franco C.M., Fente C.A., Vazquez B.I., Rodriguez J.L., Prognon P. and Mahuzier G. (1996). Post column excitation of aflatoxins using cyclodextrins in liquid chromatography for food analysis. J Of Chrom A. 721. 69-74. 9. Garner R.C., Whattam, M.M. , Taylor, P.J.L. and Stow M.W ( 1993) Analysis of United Kingdom purchased spices for aflatoxins using immunoaffinity column clean up procedure followed up by high-performance liquid chromatographic analysis and post-column derivatization with pyridium bromide per bromide, J. Chromatography, 648: 485-490 10. Blesa J., Soriano J.M., Molto J.C., Marin R. and Manes J. (2003). Determination of aflatoxins in peanuts by matrix solid-phase dispersion and liquid chromatography. J Of Chrom A. 1011. 49-54. 11. Garden S.R. and Strachan N.J.C.(2001). Novel colorimetric immunoassay for the detection of aflatoxin B1, Anal Chim Acta, 444. 187-191. 10

12. Braitina Kh. Z., Malakhova N.A. and Stojko Y. (2000) Stripping voltammetry in environmental and food analysis, Fresenius J Anal Chem 368: 307-325 13. Fifield F.W. and Kealey D (2000) Principle and practice of analytical chemistry 3rd Edition, Blackwell science, UK 14. Yaacob M.H., Mohd Yusoff A.R. and Ahmad R. (2003). Cyclic voltammetry of AFB2 at the mercury electrode. Paper presented at Simposium Kimia Malaysia ke 13 (SKAM-13), 9 – 11th September 2003, Kucing, Sarawak, Malaysia. 15. Standard procedure for determination of aflatoxins (2000). Chemistry Department, Penang Branch, Ministry of Science, Technology and Innovation (MOSTI), Malaysia (unpublished paper). 16. Smyth M. R., Lawellin D. W. and Osteryoung J.G (1979) Polarographic Study of Aflatoxin B1, B2, G1 and G2: Application of Differential-pulse Polarography to the Determination of Aflatoxin B1 in Various Foodstuffs, Analyst, 104: 73-78 17. Sabry S.M. (1999). Adsorptive stripping voltammetric assay of phenazopyridine hydrochloride in biological fluids and pharmaceutical preparation, Talanta 50: 133-140.

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