Determination of Calcium and Magnesium in [PDF]

Determination of Calcium and Magnesium in Serum, Urine, Diet, and Stool by Atomic Absorption Spectrophotometry Elizabeth G. Gimblet, Amy F. Marney,

and Roy W. Bonsnes

Atomic absorption spectrophotometry was evaluated as a method for the determination of calcium and magnesium in serum, urine, diet, and stool, and was found a most suitable technic for a large clinical chemistry laboratory.

was developed by Walsh and his associates (1) as an analytical technic applicable to the determination of many metals. Willis (2-4) found that this technic was applicable in particular to tile determination of calcium and magnesium in Serum and urine. These published results indicated that atomic absorption spectrophotometry would be a simple, rapid, and practical method for tile determination of calcium and magnesium in biologic material, particularly in serum and urine, and that it could be adapted effectively to the needs of a clinical chemistry laboratory. After evaluation, we have used atomic absorption spectrophotometry for routine determinat ion of calcium and magnesium iii serum and urine since November 1962. Since our studies of this technic began, several reports have appeared on the determination of calcium and magnesium in biologic materials (5-12), indicating considerable variation in tile methods used. Thus, it seems appropriate to publish the procedures we have found useful for routine determinations of these elements, not only in serum and urine, but also in diet and stool. ATOMIC

ABSORPTION

Instrumentation The instrument the Perkin-Elmer

used in this work, and still in routine operation, is Model 214 atomic absorption spectrophotometer (13),

From the Laboratory for Clinical Cimemistry, Time New York Hospital, and the Chemistry Laboratory of the Department of Obstetrics and Gynecology, The New York Hospital and Cornell University Medical College, New York, N. Y. 10021. Some of the data on the determination of serum magnesiimmn are from a dissertation in 1962 by the senior author in partial fulfillment of the requirememmts for the degree of Master of Arts, Hunter College, New York. Received for publication Sept. 2, 1966; accepted for publication Oct. 27, 1966. 204

Vol. 13, No. 3, 1967

205

CALCIUM AND MAGNESIUM

connected to a Sargent Model SR recorder. All data recorded were collected with tile burner described by Slavin (14). A lean acetylene-air flame has beell used for all analyses. The air compressor used supplies oil-free air. Both calcium and magnesium are determined with the use of hollow cathode lamps. The 4226-A calcium line and the 2852-A magnesium line are isolated with the monochromator of the instrument set to a spectral slit width of about 300 . The settings of the instrument for the burner, the hollow cathode lamps, and the external recorder depend upon the particular components used. Some details on these components with respect to the determination of calcium are reported by Slavin et al. (15). In general, the difficulties encountered with the instrument, including the burner, have been no greater than those an experienced laboratory worker might expect with any laboratory instrument or apparatus of equal complexity. Operation of tile instrument ilas been largely troublefree. The slot of tile burner, which is 0.020 in., tends to become constricted slightly when sera are analyzed. Currently, 27 ± 9 sera are analyzed for calcium and roughly 2 ± 2 for magnesium each day the instrument is in use. Under these operating conditions and Witil tile diluents described, the burner is cleaned once a day. How many more sera could be analyzed without tile burner’s requiring cleaning remains to be determined.

Standards and Samples Calcium carbonate (Mallinckrodt’s Primary concentrated IiCl served as tile stock standard solution was diluted appropriately to obtain made up to contain the equivalent of 2.5, 5.0, mg./100 ml. in a 1 :10 dilution of serum. Magnesium

turnings

(Mallinckrodt)

dissolved

Standard) dissolved in for calcium. This stock the working standards, 7.5, 10.0, 12.5, and 15.0 in

concentrated

HC1

served as the stock standard for magnesium. This stock solution was diluted to give working standards equivalent to 0.5, 1.0, 2.0, 3.0, and 4.0 mg./100 ml. magnesium in a 1 :25 dilution of serum.

Methods and Results Calcium In Serum

To measure the accuracy with which calcium could he determined iii serum and urine by atomic absorption speetroscopy, both Comparison and recovery studies were carried out. For serum calcium, Willis (2) deproteinized the serum with trichioroacetic acid. He also reported that

206

GIMBLET ET AL.

Clinical

Chemistry

reasonably accurate results could be achieved when serum was simply diluted with either water or 1% (w/v) EDTA. To achieve maximum accuracy with only water as the diluent, he found careful flame adjustment necessary. Since we were seeking the simplest procedure, requiring the least manipulation and the fewest steps, we used 1% EDTA as the diluent. To determine serum calcium, 0.5 ml. of serum is diluted with 4.5 ml. of 1% EDTA. With this diluent, 100.1% of calcium added to serum (5 samples) was recovered. The coefficient of variation (V) was 1.6%; the standard deviation of the sample(s) was 1.62; and s2 was 2.62. These values compare favorably with the 100.1% recovery reported by Willis, with V of 1.9% (calculated by us). To test the relative accuracy of the method further, serum calcium was determined on sera which had been submitted for routine analysis and which constituted a representative sample of abnormal sera; values obtained by atomic absorption were compared with those obtained by oxalate precipitation and permanganate titration as described by ClarkCollip (16) (a modification of the Kramer-Tisdall method (17) for the chemical determination of calcium). If hotil methods measure serum calcium with the same accuracy, tue value obtained by atomic absorption spectrophotometry (y) sllonld equal the value obtained by the oxalate-permanganate method (x). With perfect correlation the best straight line to fit the data would have a slope of 1.0 and an intercept of 0. A series of 56 determinations was run by both methods, on 10 different days. The number of determinations per (lay was or fewer on 7 days, and 8, 9, and 13, respectively, the remaining 3 days. To reduce the amount of calculation, 28 of the parallel sets of determinations (or every other one, beginning with the first), were used for statistical evaluation. Tile straight line wllich best fit these data is described by the equation y = 0.35 + 0.93 x, where y is the value obtained by atomic absorption for serum calcium and x the value by the oxalatepermanganate procedure. The deviation of the slope from 1.0 and the intercept from 0 is not significant, since t is equal to 1.363 and t is equal to 1.267, the critical value of ti% with 25 degrees of freedom being equal to 2.787. The standard deviation s is 0.345. There is no significant difference between these data and those of Willis (.2). These statistical data are presented in Table 1; the statistical parameters on Willis’ data have been calculated by us. The differences between the individual determinations in tile whole set of 56 parallel determinations have been evaluated in another way. It is assumed that the oxalate-permanganate method gives the correct

CALCIUM AND MAGNESIUM

Vol. 13, No. 3, 1967 Table

1. SERUM

CAlciuM

VAI.uEs:

(HEMIcA!,

AXE)

207

A’rOMIc

ABSORPTION

(AAS)

METhODS

Met 1,0(18

limierVo.

V

ept

PRESENT

(‘hemim.

8

1.

lb

0.345

1.565

1.267

2.056

0.016

3.185

INVESTIGATION

AAS-

EDTA

28

0.55

0.953

WILLIS

AAS0

(‘hietmi. AASEDTA

.5

AAS0

*Serurn

22

(2)

0.14

1.006

0.663

0.385’

0.25

1.013

1.788

0.124

2.086

-

deproteinized.

value; then percentage

the value obtained of this value. With

by atomic absorption this approach, the

is calculated values obtained

as a by

atomic absorption average 101.3%, or 1.3% higher than the values obtailled by the oxalate procedure (Table 2). Tile standard deviation of the differences is equal to 4.1%. The value for the significance of the difference between the two methods is 2.210. With 55 degrees of free(tom the critical value of t5% is 2.0002 and of tl% 2.664. There is thus some possibility that by atomic absorptiol1 using 1% EDTA diluent the values for serum calcium are somewhat higher than those obtained by the Clark-Collip method. This difference is of no practical significance, however. In Table 3 a more extensive set of data is shown, comparing serum

Table

2.

CALCIUM

DETERMINATION No.

BY ATOMIC

ABSORPTION,

Recovery

(%)

SERUM

This

study

Willis

5 9 56 5

()

This study Willis (2)

Coi PARED Accuracy

WITH

CHEMIC.\L

METHODS V

CALCIUM

100.1 100.0

-

101.3! 99.6!

-

URINARY

-

1.60 1.93 4.43 5.42

OLCItM

-

This study Willis (4) This study

17

99.6

5.15

Willis (4)

15

100.71

7.45

Percentage tPercentage

4 4

in comparison in

comparison

96.5 100.8

w-itli time oxalate gravimetric with the oxalate-permaaganate

3.96

1.55

method. method.

208 Table

GIMBLET ET AL. 3. SERUM

CALCIUM

BY ATOMIC

ABsORpTION

Clinical

AS COMPARED

WITII

Chemistry

OXALATE-PF.RMANGANATE

PROCEDURE

No.

Serum

1e8t3

(ml.)

38 37

8

Diluent

±3.3 ±3.8

100.1 101.0

4.5 ml., 1% EDTA 4.8 nil., 1% EDTA

0.5 0.2

calcium determinations by atomic absorption with those by the oxalatepermanganate method. The precision with which calcium can be determined, both by atomic absorption using 1% EPTA as the diluent and by tile classical chemical method, is shown in Table 4. V is quite small for replicate determinations performed the same day, and is of the same order for both the instrument procedure and the classical chemical procedure. As might be expected, precision calculated from data collected over several days with a pooled serum as a sample is not as good. The V’s for both methods are approximately 2.5 times as great as those obtained with replicate determinations performed tile same day. A V of this order with the analyses performed on different days represents a quite acceptable degree of precision for this type of analysis. In this regard, it is of illtereSt to compare these coefficients of variation with those obtained for sodium and potassium by flame photometry with the Baird Associates flame photometer (18). These data are shown in Table 5. V for sodium and potassium, as well as for serum calcium by the oxalate-permanganate method, was calculated from data obtained on pooled sera used in our routine quality control program. Since we adopted atomic absorption spectrophotometry as the routine procedure for serum calcium, it has been checked on two different occasions by the classical procedure of oxalate precipitation and perTable

4. CALCIUM

PRECISION

BY AAS

AND

OXALATE-PERMANGANATE

METHODS

Replication Concentration Method

AAS AAS AAS AAS AAS AAS AAS OX-P AAS OX-P

Solution

Standard Standard Standard Standard Standard Stammdard Serum Serum Serum Serum

(mg.I100

2.5 5.0 7.5 10.0 12.5 15.0 9.3 9.6 9.4 9.2

ml-)

Day

No.

V

Same

4 5 5 5 5

1.25 0.35 1.11 0.49 0.42 0.80 0.55 0.63 2.30 2.66

Same Saimie Same Same

Sammie Same Same Different Different

5 6 6 10 10

Vol. 13, No. 3, 1967

Table

5.

CALCIUM

PRECISION

FOR SODIUM

AND

AND

POT.tssluM

Concentration

Sodium Sodiumn Sodium I’otassiumn Potassium Potassium

IN SERUM No.

(mEqIL.)

!,ialysis

on different

BY

FLAME

PHOTOMETRY

of

V

Replicates8 5 9 8 6 8 8

141 139 137 4.6 4.8 4.4

8Amialyzed

209

MAGNESIUM

2.58 1.42 2.63 3.17 1.37 2.27

days.

manganate titration. in both instances essentially the same values for accuracy were obtained as those above: the chemical procedure yielded results equal to 98.4 and 97.3% of those obtained by atomic absorption spectroscopy. In Urine

For the determination of calcium in urine, Willis found lanthanum chloride-a 1% (w/v) solution-to be the best diluent of several tried. This diluent seemed to eliminate the interference from phosphate better than did EDTA or strontium chloride. In preliminary studies with 1% EDTA as the diluent (Table 6), recovery of calcium added to urine appeared better than when 1% lanthanum chloride was used. However, upon more comprehensive testing, dilution with 1% lanthanum chloride (see Table 7) resulted in better agreement with the chemical procedure Table

6.

RECOVERY

OF CALCIUM

ADDED

TO URINE

Recovery Dii uent

EDTA LaCb SrCl2

Table

Test8

(%)

4 4 5

97.4 96.5 90.4

7. ATOMIC

ABSORPTION

V

S

3.62 14.62 31.3

VS. CHEMICAL

1.90 3.82 5.58

METHODS

1.95 3.96 6.18

FOR URINARY

CALCIuM

Ratio .fethods

eompared*

AAS/Ox.G AAS/Ox-G AAS/Ox-P AAS/Ox-P AAS/Ox.P AAS/Ox-P 8Ox-G the Taussky

Diluent

Tests

LaCb

7 7 6 4 7 7

EDTA LaCI3 La18 LaOS3 EDTA

indicates method

time chemical (?O).

oxalate,

(%) 97.9 82.6 96.8 100.8 101.4 83.0 precipitation,

V

14.23

3.78

3.86

31.72 33.42 18.61 30.7

5.62 5.78 4.31 17.5

5.80 5.73 4.25 21.08

-

-

immcimierimtion, gravimetric

-

method;

Ox-P,

210

GIMBLET ET AL.

Clinical

Chemistry

than did dilution with EDTA. Thus our final method utilized a dilutioll with 1% lanthanum chloride. Two different methods were used for the ciieniical determination of urinary calcium. One is the classical macrogravimetric procedure in which calcium is precipitated as tile oxalate, isolated, mcmerated, and finally weighed as the oxide. This procedure was performed for us at another laboratory (19). The other method is that described by Taussky (20), which is essentially an oxalate precipitation-permangallate titratioii procedure applied after the urine has beell extracted with chloroform. In our own evaluation trials of Tanssky ‘s InetilOd, we were able in 7 trials to recover 96.3% of the calcium added to uriiie. rf he coefficient of variation was 3.06. Recovery data and accuracy tests for atomic absorption versus chenlical analyses are reported in Table 2, with comparable data from Willis (4). In Diet and Stool

The diets analyzed were duplicates of those fed to volunteer pregnant patients who were subjects for a metabolic investigation. Tile diets were planned to be adequate for optimum nutrition during pregnancy. The stools were from the same patients. For analysis, diets are first homogenized; samples of tile homogenate are placed in weighed platinum dishes and then w-eighed. They are partially dried at 90#{176}, then dried completely at 130#{176}. The dried material is ashed at 550#{176} in a Thermolyne 2000 furnace. When cool, the ash is dissolved in a few drops of nitric acid and transferred to a 23-mi. volumetric flask containing distilled water; it is then brought to volume with distilled water and appropriate dilutions made. Stools are collected in a No. 5 Lily tub lined with an 8- by 4- by 18-in. polyethylene bag, which weighs about 7 gm. The stool is frozen in this bag, and the frozen stool and bag are weighed together; thus tile weight of the stool is obtained. The frozen stool separates easily and cleanly from the polyethylene bag if it is necessary to i’emove it. In our nietabolic studies, stools from a 48-hr. period are usually combined. To the combined stool as passed, 800 ml. of water is added. Tile siligle plastic bag containing tile frozen combined stools is then encased in two more plastic bags of the same size. The bags are closed by twisting the open ends together, then folding tile twisted ends over. Closure is maintained with a heavy rubber band wrapped around the folded-over ends. This package is placed in a 1-gal. paint can and the cover is closed. Tile can is then shaken for 15 mm. on a paint mixer. A small sample of the re-

Vol. 13, No. 3, 1967

Table

CALCIUM

8. RECOVERY

OP CALCIUM

AND

ADDED

211

MAGNESIUM

TO DIET

AND

STOOL Total

Somnie

(‘a in Saom,mle*

mit. (ym.)

HOMOGENATES

(MEQ)

Ca Recovery

(a

added

Expecied

Found

(% ) t

0.241 0.200 0.245 0.209

100.8 100.5 102.1 96.8

0.463 0.515 0.425 0.438

97.2 100.6 100.2 97.3

DIET HOMOGENATE

5.2658 4.1320

0.184 0.144 0.185 0.161

5.3061 4.6150

0.055 0.055 0.055 0.055

0.239 0.199 0.240 0.216

STOOL HOMOGENATE

5.3295

0.421

5.7858 4.6709 4.9942

0.457 0.369 0.395

0.055 0.055 0.055 0.055

5By

0.476 0.512 0.424 0.450

amialysis, 1 gill. of diet was foumid to 0.079 ,miEq. of Ca. tAverage; for diet, 100,1%; for stool, 98.8%.

comitain

0.034

mmmEq. Ca,

and

1 gum. of

stool

liclliogenate,

sultiiig homogenate is then weighed in a preweighed dish. The remainder of the sample preparation is the same as that t’or diet. Studies of the recovery of calcium added to diet and stool are shown in Table 8. These data indicate the accuracy of the over-all process. For the final dilution, 0.5 ml. of the diluted solution to be analyzed is added to 4.5 ml. of 1% lanthanum chloride. The diets and stools analyzed in tins study are not necessarily representative of all possible diets or stools. Analysis of stools from patients receiving antacids like Amphojel,* which contains aluminum salts, may be subject to error because of tile presence of these salts in the stool. Magnesium

To measure tile accuracy with which magnesium can be determined in serum and urine by atomic absorption spectrophotometry, only recovery experiments, constituting the so-called internal-standard method of evaluation, were employed. In the procedure usually used for serum magnesium, 0.2 ml. of serum was diluted with 3 ml. of diluent. As shown in Table 9, we have found, in agreement with the observations of Willis (3, 4), an enhancement of absorption wilen water is used as a diluent and acceptable recoveries of added magnesium when 0.25% strontium chloride is used as a diluent. Thus tile strontium chloride seems to suppreSS served *Wyeth

tile

enhancenient.

when

15%

Laboratories,

TCA

A similar was used

Philadelphia,

Pa,

enliancenient to precipitate

of absorption the protein.

was This

ob-

effect

212

IMLET Table

9.

RECOVERY

T AL.

OF MAGNESIUM

ADDED

Clinical TO SERUM

AND

Chemistry

URiNE

Recovery Diluent

(%)

Tests

Serum SrCl3 H,O TCA Urine H20 SrCI2

.

101.2 107.8 107.9

2.44 2.74 13.63

1.56 1.65 3.68

1.55 1.53 3.41

5 4

99.4 101.1

8.31 9.17

2.88 3.03

2.90 3.00

OF MAGNESIUM

FROM

DIET

AND STOOL HOMOGENATES Total

Sample

wt.

M9 in

(gm.)

V

4 4 4

10. RECOVERY

Table

5

(MEQ)

Mg

Mg

sampie*

Recovery

added

Expected

(%)t

Found

DIET HOMOGENATE

4.8731

0.058

0.028

0.086

0.083

4.7229

0.057

0.028

0.085

0.083

4.9651

0.060

0.028

0.088

0.087

99

3.6761

0.044

0.028

0.072

0.073

101

103.9 100.0 100.5 106.1 98.1

HOMOGENATE

STOOL

5.4884 5.6433

0.178 0.183

0.027 0.027

0.205 0.210

0.213 0.210

4.9010 4.4241 6.5505

0.159 0.143 0.212

0.027 0.054 0.054

0.186 0.197 0.266

0.187 0.209 0.261

3By 0.0324

analysis, mEq.

Average:

Table

11.

1 gm.

of diet

was

97 98

found

to contain

0.012

mEq.

of Mg,

and

1 gm.

of

stool,

Mg. for diet,

99%;

COEFFICIENTS

for

OF

stool,

VARIATION

Concentration

101.6%.

FOR No.

STANDARDS,

REPLICATED

MAGNESIUM

BY

ATOMIC

ABSORPTION

V

of replicates

ON SAME

DAY

(IG./100

MI..)

0.5

5

1.06

1.0

5

0.56

2.0

5

1.05

3.0 4.0

5 6

0.34 1.64

SERA;REPLICATRD

1.82 1.82 l2

ON DIFFERENT

5 5 16

METHOD

DAYS

(MEQ/L.)

.

1.10 2.07 2.52

Vol. 13, No. 3,

Table

967

CALCIUM

12. RECOVERY

OF

Urine dilution

Mg in urine

213

MAGNESIUM

ADDED TO URINE

MAGNESIUM OR WATER

AND

AS DILUENT Mg added

WITH

(MG./100 Total Mg

STRONTIUM

0.25%

STRONTIUM

CHLORIDE

ML.) Mg found

Recovery

(% I *

CHLORIDE

1 to 100

0.64

0.5

1.14

1.17

103

2 to 100

1.34

0.5

1.84

1.86

101

3 to 100 4 to 100

1.96 2.66

0.5 0.5

2.46 3.16

2.55 3.06

104 97

0.51 0.63 1.23

0.49 0.63 1.23

96 100 100

WATER

0.4 to 100 0.5 to 100 ito 100

0.31 0.38 0.73

0.2 0.25 0.50

2 to 100

1.43

0.50

1.93

1.88

98

3 to 100

2.0

0.20

2.20

2.28

104

5Average:

with

strontium

chloride,

101C% ; with

water,

100%.

observed whether strontium chloride was present or not. The diltient used routinely for serum magnesium determination is 0.23% stroiitium chloride. Recovery of magnesium from diet and stool honiogenates is shown in Table 10. The final solutions used in these analyses consisted of 0.1 ml. of appropriately diluted ash solution in 5.0 ml. of water. The precision with which magnesium can be detei’rnined is shown in Table 11. Again, as expected, the precision obtainable is much better when the replicate analyses are performed on the same day than when they are performed on different days. Table 12 shows the recovery of magnesium from urine with 0.23% strontium chloride and with water used as diluelsts. Water as the diluent appears to give slightly better results statistically, but the difference is not significant. For the sake of simplicity, water is used as the preferred diluent. was

References 1. 2. 3.

4. 5. 6.

Walsh, A., The application of atomic absorption spectra to chemical analysis. Spcclrochim. 4cta 7, 108 (1955). Willis, .J. B., The determimmatioim of metals in blood serum by atomic absorptiomm spectroscopy. I. Calcium. Spectrochim. Acta 16, 259 (1960). Willis, J. B., The determination of metals in blood serum by atomic absorption spectroscopy. II. Magnesium. Spcetroehim. Acta 16, 273 (1960). Willis, J. B., Determination of calcium and magnesium in urine by atomic absorption spectroseopy. Anal. Chem. 33, 556 (1961). Newbrun, E., Application of atomic absorption speetroseopy to time (letermlminatiOn of calciummmin saliva. Nainre 192, 1182 (1961). Dawson, J. B., and Heaton, F. W., The determination of magnesium in biological materials by atomic absorption spectrophotometry. Biochem. J. 80, 99 (1961).

214 7.

8.

9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

GIMBLET fT AL.

Clinical

Chemistry

Herrnmanmm, R., and Lang, W., Analysen vomi Magmiesiumn imi Serum umid im anderen K#{246}rper flussig Keiten mit Hilfe der Absorptions flammen Potometre. Z. Ges. Exp. Med. 135, 569 (1962). Somne biological applications of atomic absorption spectrophotomnetry. Atomic Absorption Newsletter No. 11, March 1963. Horn, D. B., and Latner, A. L., The estimnation of magnesium by atomic absorp’tion spectropliotomnetry. C/in. C/mini. Aeta 8, 974 1963). Stewart, W. K., Hutehmimison, F., and Fleming, L. w., The etimnation of magimesium in Serum alid urine by atomnic speetrophotometmy. J. Lab. C/in. Med. 61, 858 (1963). Decker, C. IF., Aras, A., and Decker, L. B., Determination of magnesium amid calcium iii cerebrospinal fluid by atomic absorption spectroscopy. Anal. Bioc/tein. 8, 344 (1964). Zettmmer, A., and Seligson, D., Application of atomic ahsorptioim spectrophotomnetry in the determimiation of calcium in serum. Gun. Chew. 10, 869 (1964). Leen, M. W., and Atwood, J. G., Design of an atomic absorption speetrophotometer. Pittsburgh Conference Oil Analytical Chemistry, Feb. 27-Mar. 3, 1961. Slavimi, W., A burner-atomizer for atomic absorption speetrophotometry. Atomic Absorption Newsletter No. 10, February 1963. Slavin, W., Sprague, S., and Manning, D. C., The determination of calcium by atomic absorption spectrophotometry. Atomic Absorption. Newsletter No. 15, September 1963. Clark, E. P., and Collip, J. B., A study of the Tisdall method for the determination of blood serum calcium with a suggested modification. J. Biol. Chew. 63, 46i (1925). Kramer, B., and Tisdall, F. IF., A simple technique for the determimiation of calcium and magnesium in small amounts of serum. J. Biol. Ch.em. 47, 475 (1921). White, J. U., Precision of a simple flame photometer. Anal. Chem. 24, 394 (1952). Wolfson, A. H., and Meyer, W., Time detcrniination of calcium iii urine. Personal communication.

20.

Taussky, H. Microchem.

H., Microdeterimiinatioim J. 7, 89 (1963).

and

separatiomi

of

calcium

and

strontium

in urine.