Idea Transcript
Shawn P. Vorce Technical Director Chesapeake Toxicology Resources
"The opinions or assertions presented hereafter are the private views of Shawn P. Vorce and should not be construed as official or as reflecting the views of the Department of Defense, its branches, the US Army Medical Research and Materiel Command or the Armed Forces Medical Examiner System.“
Forensic Toxicology is defined as the application of toxicology for the purposes of the law Postmortem Toxicology Death investigations Cause and manner
Human Performance Toxicology DUI/DWI DFSA
Forensic Drug Testing
Military Criminal Justice System Workplace drug testing Athletic programs
negative
Accessioning
Special Testing
Screening
Method Development
positive
Issue Report
Consultation/Testimony
Confirmation/Quantitation
Screening ◦ ◦ ◦ ◦ ◦
Immunoassay (IA) Thin Layer Chromatography (TLC) Headspace (GC/FID) Gas Chromatography (NPD/FID) Gas Chromatography/Mass Spectrometry (GC/MS)
Confirmation/Quantitation
◦ Gas Chromatography/Mass Spectrometry (GC/MS) Full Scan Selected Ion Monitoring (SIM) ◦ Liquid Chromatography/Mass Spectrometry (LC/MS) ◦ Liquid Chromatography Tandem Mass Spectrometry (LC/MS/MS) ◦ Time of Flight Mass Spectrometry (TOF)
Immunoassay Screening ◦ EMIT enzyme multiplied immunoassay technique
◦ CEDIA
cloned enzyme donor immunoassay
◦ ELISA
enzyme-linked immunosorbent assay
◦ FPIA
fluorescence polarization Immunoassay
◦ KIMS
kinetic interaction of micro-particles in solution
◦ DRI
Diagnostic Reagent Inc
Immunoassay Screening ◦ EMIT
Sample, Antibody, Enzyme-drug complex, Substrate
substrate
Sample
Does NOT Absorb light
Drug
Drug
Drug
substrate
Sample Enzyme-drug complex
Absorbs light λ= 340 nm
Immunoassay Screening
Positive Result Negative Result
IA Cross Reactivity
Benzodiazepines (CEDIA) ◦ Oxazepam antibodies ◦ 200 ng/mL cut-off
◦ Package insert list interfering compounds and levels
IA Cross Reactivity Amphetamines (CEDIA) ◦ Amphetamine antibodies ◦ 500 ng/mL cut-off
Compound
500 ng/mL Cut-off
Bupropion
13000
Fenfluramine
1200
Phenmetrazine
1500
Phenothiazines
10000
◦ Package insert list interfering Phentermine compounds and levels Pseudoephedrine
17500 75000
DMAA
2500
Methamphetamine
500
MDA
1400
MDMA
800
Headspace-GC/FID
BAC ◦ Headspace Sample and ISTD are transferred to a headspace vial Vial is heating to a constant Temp (90oC). Volatile component diffuse into gas phase forming equilibrium between liquid and headspace. Sample is taken from the Headspace and analyzed.
Headspace
BAC
◦ Headspace
http://blog.restek.com/wp-content/uploads/2014/05/HS-GC-Instrument-Diagram2.jpg
BAC
Acetone ISTD
2.00 1.50
Acetaldehyde
0.50 1.00
2-Propanol
0.0
Ethanol
2.50
3.00
◦ Headspace (GC/FID) Ethanol Response/ ISTD Response
0.0
0.02
0.05
0.08
0.10
0.12
Ethanol Concentration
= 0.088 BAC
0.15
Inhalants 1.615
0.883
◦ Headspace (GC/MS)
Standard Volatile Profile
1.116
1.857
200
Unidentified Peak 2.985
120
100
2.986
1.742
150
160
80
50
0.823
0.823
40
0.5
1
1.5
2
2.5
3
3.5
min
0.5
1
1.5
2
2.5
3
3.5
Inhalants
0.883
◦ Headspace (GC/MS)
51
160
100
Unidentified Peak 2.985
120
F
50
F
65
80
47 45 27 26 31 33
15 2
0
0
10
20
30
40
46
64
43
40
0.823
67
50
60
(mainlib) Ethane, 1,1-difluoro-
70
80
0.5
1
1.5
2
2.5
3
3.5
Screening
◦ Thin Layer Chromatography (TLC) Spot •sample •standards
develop
Dry plates in an oven
Spray
Gas/Liquid Chromatography
Gas/Liquid Chromatography
Inject Sample
Fused Silica Capillary Column
Temperature Programmable Oven
Detector
2.216
200
3.756
Chromatogram 5.991
3.511
150
100
50
1.0
2.0
3.0
4.0
5.0
6.0
7.0
min
Screening
◦ Gas Chromatography (GC/FID or GC/NPD)
Positive Identification RT Library Automated Search Criteria: RT ±2% of STD
Mass Spectrometry
a-methyltryptamine N
F3CF2CC
Abundance 12500 12000 11500 11000 10500 10000 9500 9000 8500 8000 7500 7000 6500 6000 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0
m/z-->
O
276
N
F3CF2CC
O
303 H N
N
129
O C CF2CF3
190 H N
N
F3CF2CC
69
O
102 156
466
51
256 40
60
80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460
O C CF2CF3
Screening/Confirmation
◦ Gas Chromatography/Mass Spectrometry (GC/MS) L
Abundance 360000 340000 320000 300000
280000 260000 240000 220000
N
200000
D
V
180000
M
160000 140000
H
C
120000
K O
100000 80000 60000 40000
A
F
B
E
20000
Time-->
0
5.00
6.00
7.00
8.00
Q
I G
J
9.00
P 10.00
R
T
U
S
W 11.00
12.00
Figure 7 Full scan EI total ion chromatogram of 27 designer phenethylamines and tryptamines. (A) 4-FA (B) 4-FMA (C) 4-MA (D) 4-MTA (E) DOM (F) DOET (G) 3,4,5trimethoxyamphetamine (H) 2C-C (I) DOC (J) 5-IT (K) AMT (L) DMT (M) 2C-B (N) DOB (O) 2C-T (P) 2C-I, DOI (Q) 2C-T-2 (R) 5-MeO-AMT (S) 5-MeO-DMT (T) DiPT (U) 5-MeO-MiPT (V) 5-MeODiPT (W) 5-MeO-DALT.
Screening/Confirmation
◦ Gas Chromatography/Mass Spectrometry (GC/MS) ◦ Full Scan MS
Positive Identification
Urine Sample
Mass Spectral Library Automated Search Criteria: RT ±2% of STD Match of >70% screen Match of >90% confirm
No extraneous ions
Standard Solution
MS Full Scan Library Match
Confirmation/Quantitation
◦ Gas Chromatography/Mass Spectrometry (GC/MS) Full Scan Spectrum at 11.25 min
Cycle: Scan 40-550 m/z
7 Cycles Library Match
Confirmation/Quantitation
◦ Gas Chromatography/Mass Spectrometry (GC/MS) Selected Ion Monitoring (SIM)
SIM Cycle: 303, 182, 82 m/z
16 Cycles Improves peak shape, sensitivity, quantitations
Confirmation/Quantitation
◦ Liquid Chromatography/Mass Spectrometry (LC/MS)
Confirmation/Quantitation
◦ Liquid Chromatography Tandem Mass Spectrometry (LC/MS/MS)
LC/MS/MS
MRM Transition 342.0 / 155.1
Product Ion Scan (Full Scan)
MRM Scan
Full Scan
Monitor specific Compound at Expected RT 750 compounds in 12.0 min
Waters Xevo G2 QTOF AB Sciex 5600+ TripleTOF
Ion Mirror
Shield (“Liner”)
10-7 Torr
Turbo Pump B Turbo Pump A
Rough Pump
1 Torr
10-5 Torr
10 mTorr
Shroud
10 mTorr
Shroud
Shroud
1
2
DC1
DC2
1
2
Ion Focus
Accelerating Column
Upper Slit
MCP Detector
Slicer Puller
Fragmenter DC
Pusher Skimmer 1
a Lens 1 and 2
Collision Cell (CC or Q2)
b Exit Pulse
Time of Flight = m/z
Lower Slit
Measure Current for Beam Alignment
Octupole 1
Quad Mass Filter (Q1)
Octupole 2 Collision Gas (Ar)
Version 20040304
DC Quad (Shaping Quad or QDC)
Time of Flight (TOF)
Nominal mass Exact (calculated) mass
300 300.1594
H+ CODEINE [M+H]+
300.1595
Exact Mass Libraries ◦ 700-1400 compounds pre-loaded
High resolution – (4-5 decimals) ◦ Same NOMINAL mass but different EXACT mass
Elucidate unknown peaks ◦ Propose chemical formula
“All data” acquired ◦ Reprocess archived data for new compounds
Drug 0 386 2123 4312 10732 23461
Quantitation ISTD 0 2097 2325 2220 3123 3466
6-acetylmorphine Drug Conc ISTD Conc x 0 0 0 5.0 25.0 0.2 25.0 25.0 1.0 50.0 25.0 2.0 100.0 25.0 4.0 200.0 25.0 8.0
y 0 0.1841 0.9131 1.9423 3.4364 6.7689
Calc Value 0 5.38 26.69 56.77 100.45 197.85
% Diff 0 7.61% 6.76% 13.55% 0.45% -1.07%
Unknown Drug Response ISTD Response ISTD Conc 5.29 575 3166 25.0 y-int 0
m (slope) 0.8553
8.0000 y = 0.8553x R² = 0.9977
7.0000 6.0000 5.0000
Ser
4.0000
Line 3.0000 2.0000 1.0000 0.0000 0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
Confirmation/Quantitation
Positive Identification Criteria: RT ±2% of avg Calibrators 2 Ions (ISTD) 3 Ions (Compound) Ion Ratios ±20% of avg Calibrators Gaussian shaped peaks Peak asymmetry
Confirmation/Quantitation
131
Abundance
NH 2
44
35000
30000
Full scan EI mass spectra of 5-IT and AMT
N H
5-IT C 11 H 14 N2 mw 174.25
25000 20000 15000
RT
10000
5000
77
51
103
65
174
154
115
89
0 m/z -->
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
131
Abundance
30000
5-IT
9.10 min
AMT
9.30 min
NH2
44
26000
22000
Ion Ratios
N H
18000
AMT C 11 H 14 N2 mw 174.25
14000
10000 6000
2000 m/z--> 0
77
103
52 40
50
63 60
117
89 70
80
90
100
110
120
143 130
140
159 150
160
174 170
180
5-IT 131/130 m/z
1.75
AMT 131/130 m/z
1.14
Chain of Custody
Standard Operating Procedure (SOPs)
Quality Assurance/Quality Control
Screening/Confirmation/Quantitations Methods
Case (list of item received) Each aliquot tested
Administrative, Accessioning, Safety, Testing Methods, etc. Controls
Method Validations
Specificity, selectivity, accuracy, precision, carryover, stability, variation, robustness, etc.
Instrumentations
Tuning Calibration System Suitability Controls
Criteria for Positives/Confirmation ◦ Retention Times (RT)
GC ±2% of avg Calibrators LC ±3% of avg Calibrators
◦ Full Scan MS
>70% screen >90% confirmation
◦ SIM
2 Ions (ISTD) / 3 Ions (Compound of Interest) Ion ratios ±20% of avg Calibrators
◦ MRM Ion Transitions
2 MRMs (ISTD or Compound of interest) MRM Ratios ±20% of avg Calibrators
◦ ToF
LC ±3% of avg Calibrators Exact mass ±5 ppm
◦ Peak asymmetry 0.5 – 2.0