Determination of Calcium in Urine and Serum by ... - Clinical Chemistry

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precipitation, ashing to carbonate, dilution. 1:50 in. 0.1% lanthanum, and determination of calcium concentration by atomic absorption spectrophotometry. Thus,.

Determination of Calcium in Urine and Serum by Atomic Absorption Spectrophotometry (AAS) David L. Trudeau

and Esther F. Freier

The determination of calcium in biologic fluids by atomic absorption spectrophotometry is interfered with by the presence of protein, cations, and those anions that form complexes with calcium. Such interference was overcome when lanthanum was included in a 1:50 dilution of serum or urine. Recovery of calcium added to calciumfree serum was 100%. The S.D. based on double-blind duplicates was 0.22 mg./lOO ml. Excellent statistical agreement was found between the test method and each of the 2 reference methods.

N

have been proposed for the measurement of calcium in biological materials. These methods have utilized oxalate precipitation followed by permanganate or acidometric titration (1, 2), direct chelatometric titration of calcium with ethylenediaminetetraacetic acid (EDTA) using one of several indicators (3-5), and emission flame photometry (6). While oxalate precipitation is generally accepted as a reference procedure, it is time-consuming and requires considerable sample volume. Chelatometric titrations are deficient in the presence of lipemia, hemolysis, and high phosphate levels, particularly the levels found in urine. Emission flame photometry, while rapid, suffers from background interference from both cations and anions. Furthermore, the existence of numerous variations of these basic methods for calcium determination implies general dissatisfaction with them. In 1960 Willis (7), using an atomic absorption flame spectrophotometer developed by Walsh (8, 9), demonstrated that atomic absorption spectrophotometry (AAS) is suitable for measuring calcium in serum. Willis suggested that removal of protein from the sample prior to UMEROUS

METHODS

From the Department of Laboratory Medicine, Minneapolis, Minn. 55455. The assistance of Professor Jacob E. Bearman tion data is gratefully acknowledged. Received for publication May 11, 1966; accepted -

101

University with

the

of

Minnesota

statistical

for publication

July

analyses 25, 1966.

Medical of the

School, correla-

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& FREIER

Clinical

Chemistry

determination is desirable, and demonstrated that interference caused by anions bound to calcium in solution could be overcome by the addition of an excess of lanthanum, strontium, or EI)TA. Zettner and Seligson (10) showed in 1964 that lanthanum added to diluted serum prevented both the depression effects of phosphate and sulfate and the depression and enhancement effects of protein on the atomic absorption of calcium. They advocated measurement of serum calcium by AAS in samples prepared by direct dilution 1 :10 iii a complex diluent of lanthanum, butanol, octanol, and HC1, using standard calcium solutions containilig protein, sodium, and a bacteriostatic agent. Sunderman and Carroll (ii) have likewise presented a method for determination of calcium in serum by AAS in which the 1 :10 protein-free filtrate offer trichloracetic acid precipitation is diluted 1 :1 with a 0.5% strontium solution. With dilutions of serum less than 1 :25, some method must be found to avoid the troublesome clogging of the burner. Zettner and Seligson (10) avoided this effect by adding organic solvents to their diluent mixture and Sunderman and Carroll (11), by removing proteins prior to burner aspiration. Because the Perkin-Elmer 303 atomic absorption spectrophotometer* enjoys some increase in sensitivity (12) over the instrument used by Zettner and Seligson (10)-a Perkin-Elmer 214-it was decided to study the interference effects on a 1 :50 dilution of serum in a simple lanthanum chloride diluent, avoiding the higher protein concentration in the 1 :10 dilution, and thereby eliminating the need for organic solvents and protein removal. Furthermore, the results of Zettner and Seligson (10) suggest that 0.5% lanthanum retards the depressant effects of phosphate and sulfate on calcium absorption so adequately that it would be a suitable diluent for the determination of calcium in urine by AAS at a 1 :50 dilution. It is the purpose of this paper to present a method for the determination of calcium in serum and urine by AAS that involves only direct dilution of sample in lanthanum solution. It is rapid and precise, and agrees well with other methods.

Materials and Procedure Lanthanum Diluent

Lanthanum and diluted num (w/v).

chloride, LaC13#{149}7 1120, was dissolved so as to make solutions that were 0.5%

Perkin#{149}Elmer Instrument

Corp.,

Norwalk,

Comm.

in distilled and 0.1%

water lantha-

Vol. 13, No. 2. 1967

Calcium

CALCIUM

IN URINE AND

SERUM

103

Standards

Pure pulverized CaCO3 was weighed to the nearest ten thousandth gram, dissolved in 1 N HCI, and diluted with distilled water to concentrations of 100.0 mg./L. and 200.0 mg./L. of calcium. A calcium standard of 150.0 mg./L. was made by mixing equal parts of these 2 standards. The 50.0 mg./L. calcium standard was prepared by diluting the 100.0 mg./L. solution 1 :1 with distilled water. Sample Preparation

Using either acidified urine in a diluent of 0.5% La+++ or serum in a diluent of 0.1% the specimens were diluted 1:50 with an 0.2-ml. Seligson pipet fitted to a 50-ml. buret. Standards of 50.0, 100.0, 150.0, and 200.0 mg./L. were then diluted in a like manner in the appropriate diluent to produce diluted standards equivalent to 5.00, 10.00, 15.00, and 20.00 mg./100 ml. of serum or urine calcium. The samples were then assayed for calcium content by atomic absorption spectrophotometry. Determination

0f Calcium Content by Atomic Absorption Spectrophotometry

A Perkin-Elmer 303 atomic absorption spectrophotometer using a Perkin-Elmer calcium cathode source was used throughout. The source was used at 10 ma. and the diffraction grating set to allow peak energy of the 422.7-mg resonance line of calcium. The scale was used at 1, the response time at 0.1, and the slit set at 0.4. Air was used at a pressure of 26 psi and acetylene at a pressure of 10 psi. The flow was adjusted so that the air-flow meter read 8#{189} units and the acetylene flow meter read 9 units. The capillary aspirator was adjusted so that a sample aspiration rate of 4.2 ml./min. was obtained. Prior to each use of the instrument the cathode was allowed a 30-mm. warm-up period. The diluted specimens and standards were then aspirated into the burner atomizer via a polyethylene tube connected to the atomizer capillary and 3 independent readings were taken of each solution on the percentage of absorption counter. The usual sequence was as follows: First, the diluent alone was aspirated and the null meter adjusted so that the null meter reading was “0” when the percentage of absorption counter read 00.00. The percentage of absorption was then determined for the 4 standards. Diluted specimens were then aspirated and percentage of absorption readings taken. Between each 3 specimen readings, a standard reading was obtained and if the standard reading varied by more than 1% absorption, the percentage of absorption of the standards was redetermined. The percentage of absorption readings were averaged, converted to absorbance, and values for calcium concentration determined either with computations or graphs. A typical standard line is seen in Fig. 1.

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Experimental Results The Effect of Sodium on Calcium Absorption

We found that 1:50 dilutions of calcium standard in water had a lower absorption than 1 :50 dilutions of the same standard with physiological amounts of sodium in water. Willis (7) has stated that sodium enhances the absorption of calcium in AAS, whereas Zettner and Selig-

Fig. 1. Typical

AAS

plot

of

flame absorbance versus calcium concentration. Calcium standard diluted 1:50 in 0.1% lanthanum.

CALCIUM

STANDARD

(mgjsOOmI.1

son (10) have said that physiological amounts have a depressant effect on calcium absorption. Trent and Slavin (13) described this effect of sodium as being one of enhancement and showed that, in the presence of 1% lanthanum, sodium added in concentrations up to 1000 ppm does not cause significant enhancement of calcium absorption. Therefore, it was decided to study the effects of varying concentrations of sodium on the absorption of solutions containing 10 mg./100 ml. of standard calcium solution diluted 1:50 in water and 1 :50 in lanthanum solution. The results of this experiment are presented in Table 1. Concentrations of sodium ranging from 1/200 to 50 times that found in serum were studied, and it may be seen that with increasing concentration there was significant enhancement of calcium absorption on the order of 10-12% greater than expected from the amount of calcium added to the solution. As the concentration of sodium increased above 3000 mEq./L. and approached the limit of solubility of NaC1, this enhancement was still present, yet became less marked. However, it was found that a 0.1% solution of lanthanum was able to compensate entirely for this enhancement effect of sodium on calcium absorption at a 1 :50 dilution over a wide range of concentration. We also found that additions of potassium in physiological concentrations to lanthanumdiluted sodium-calcium preparations did not alter absorption.

Vol. 13, No. 2

Table

1967

1.

CALCIUM

EFFECT

Op

SODIUM

IN URINE AND

cONCENTRATION

ON

AroMIc %

(on,-entratwn

prior

105

SERUM

recovery

ABSORPTION

OF CALCIUM

ralcium

VaCI

to dilution (mEqIL.)

Diluted I :50 in water

0. 0.775 1.55 3.10 7.75 15.5 31.0 77.5 155. 310. 775. 1550. 3100. 7750.

100 102 104 109 110 113 113 111 113 109 110 111 107 106

in

Diluted I :50 0.1% lanthanum

100 100 101 101 102 98 101 102 101 101 104 100 97 95

Phosphate Depression of Calcium Absorption

The effect of phosphate has been described as causing a depression of calcium absorption. This depression has been overcome by using metals, such as lanthanum and strontium, which are chemically similar to calcium. These are used in large excess, thereby causing the interfering anions to be occupied by the added metal. The ability of a 0.1% lanthanum diluent to overcome the phosphate depression that occurs in serum at a 1 :50 dilution was studied and the results are presented in Fig. 2. When water alone is used as the diluent, a marked progressive depression of calcium absorption is seeii. Added sodium in physiological concentrations fails to alter this depression, and lowering the pH of the diluted sample to pH 1 only slightly retards this depressant effect. However, if 0.1% lanthanum diluent is used, then the depressant effect of phosphate, at least in concentrations up to 20 nig./100 ml., is overcome. The ability of a 0.5% lanthanum diluent to overcome the phosphate depression that occurs in urine at a 1 :50 dilution was studied, and the results are presented in Table 2. Other Interfering Substances in Urine

In addition to the interfering effects of sodium and phosphate on the absorption of calcium in urine already mentioned, the effects of sulfate, citrate, and oxalate were also studied. These interfering substances were added to aqueous standards containing 10 mg./100 ml. of calcium

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Clinical

Cliemhfry

and these were then diluted 1 :50 in 0.5% lanthanum. The maximum concentrations of the interfering substances added are considered the upper limits of normal in persons producing 1 L. of urine per day. The prepared specimens are presented as percentage recovered when compared to the same 10 mg./100 ml. of calcium standard. Tile results are presented in Table 2, and it may be seen that no depression occurred. Calcium Recovery by Different

Methods

In order to assess several atomic absorption methods for serum calcium analysis, a calcium-free serum was employed to which known amounts of calcium were added. Calcium was removed from serum by precipitating the calcium with an excess of oxalate and then removing the excess oxalate remaining in the supernatant with excess lanthanum. For each milliliter of calcium-free serum, 1 ml. of pooled 3 ml. of 0.5% (w/v) ammonium oxalate were transferred centrifuge tube, mixed, and placed in a 500 water bath for Following centrifugation for 10 mm., the supernatant was To this was added 1 ml. of 1.8% La as aqueous LaC13.

serum and to a conical 30-60 mm. recovered. The lantha-

10

8

o,ciII(.)rnlqNacl

Phosphate Fig. 2. Effect lanthanum.

of phosphate

Expressed

on recovery

asmg%

of calcium

P

by AAS

Prior to Dilution in the

presence

and

absence

of

CALCIUM

Vol. 13, No. 2, 1967

IN URINE AND

SERUM

107

num mixture was allowed to sit at room temperature for 90 mm. and was then centrifuged for 10 mm. The resulting supernatant was thus a 1:5 dilution of pooled serum from which calcium had been removed. This supernatant contained 4.5 mg./100 ml. of phosphorus in phosphates, 6.5 gm./100 ml. of total proteins, and less than 0.1 mg./100 nil. of calcium (by atomic absorption spectrophotometry analysis), all values expressed as concentrations in undiluted serum. In each method studied the final dilution was equivalent to a 1 :50 dilution of serum containing the indicated concentration of calcium. In Table 3, the recovery of calcium in the 1 :5 dilutions of the prepared Table 2.

EFFECT

op

INTERFERING USING

SUBSTANCES LANTHANUM

ON RECOVERY

OF URINARY

CALCIUM

DILUENT*

Concentration (my./100

%

mL)t

recovery

OXALATE

100 102 103 99 99

2 4 6 8 10 P IN NaH,PO,

100 98 97 100 101 101

20 40 60 80 100 300 S IN NflSO4

100 100 100 100 99

40 80 120 160 200 CITRATE

20 40 60 80 100 *Aqueous

standards

98 101 99 100 99 containing

10 mg./100

ml. of

Ca

to which

been added were diluted 1 :50 with 0.5% lanthanum solution. tCalculation of concentration based on original Ca solution.

interfering

substances

had

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TRUDEAU

Table 3.

CALCIUM

RECOVERED KNOWN

Diluted Ca added (mg./100 ml.)

0.0 5.0 10.0 15.0 20.0

Standards

0.4 5.8 11.0 16.3 23.6

FROM

AMOUNTS in

1:50

& FREIER DILUTIONS

OF CALCIUM eater

Clinical OF CALCIUM-FREE

(MG./100

0.0 4.9 10.2 15.3 21.1

+

NaCI

AND

ML.) Diluted

Standards

SERUM

Chemistry

Standards

0.0 4.8 9.9 15.1 20.2

in

0.1%

lanthanum Standards

+

NaCI

0.0 4.7 10.2 15.1 20.2

serums further diluted 1 :10 in water is compared to standards diluted 1 :50 in water only, and to standards containing 1 :50 dilutions of physiological sodium chloride. It is seen that there is a marked enhancement when the prepared serums are read against the standards in water only, whereas the enhancement is minimal when appropriate amounts of sodium are added to the standards. Therefore, it would seem that the bulk of the enhancement is due to the presence of sodium in serum. If 1:5 dilutions of the prepared serums were diluted 1:10 in 0.1% lanthanum, so as to be equivalent to 1 :50 dilutions of serum in approximately 0.1% lanthanum, and read against standards diluted in 0.1% lanthanum, complete recovery was obtained, as may be seen in Table 3. The addition of sodium to the standard solutions had no effect on their absorption. Thus, it appeared that the method of 1 :50 dilution of serum in 0.1% lanthanum avoids the interference of sodium, phosphate, and protein in combination and is suitable for serum calcium analysis by AAS. The addition of sodium to the standard solutions was found to be completely unnecessary. In order to assess recovery of calcium from urine, a different approach was used. using 3 different methods of sample preparation, urine calcium by AAS was determined on 7 urines randomly chosen from the lot of urines that had come to the laboratory for calcium determination by the Sobel and Sobel (2) method, and which were known to contain measurable amounts of calcium. In the first method, the urine was diluted directly 1 :50 iii water; in the second, the urine was diluted directly 1.:50 in 0.5% lanthanum; and in the third, the calcium was isolated from the urine by oxalate precipitation. The urines were acidified to pH 1 with concentrated HC1 prior to sampling in order to dissolve inorganic residue amid to insure homogeneity. In the oxalate precipitation, 1 ml. of acidified urine from each specimen and 1 ml. of each of the 4 calcium standards were transferred to centrifuge tubes. One milliliter each of distilled water and saturated ammonium oxalate were then added to each tube and the contents were

Vol. 13, No. 2, 1967

CALCIUM

IN URINE AND

109

SERUM

well mixed. Precipitation was allowed to proceed for at least 30 mm. at 560, the precipitate was recovered by centrifugation, dried and ashed in a muffle furnace for at least 3 hr. at 500#{176}. The CaCO3 residue was then dissolved with 1 drop of concentrated HC1, quantitatively transferred to a 50-mi. volumetric flask, and diluted with 0.1% lanthanum. As may be seen in Table 4, when the urine was diluted in water, lower values were obtained. When diluted directly in 0.5% lanthanum, the values obtained were identical to those obtained when the calcium was isolated from the urine by oxalate precipitation. The method of direct dilution of urine in 0.5% lanthanum thus gave results in good agreement with a method that avoids all of the possible interfering substances in urine that are known to interfere with calcium absorption. Precision

The standard deviation for blind duplicate analysis of serum calcium by direct dilution in 0.1% lanthanum and determination of calcium by AAS was calculated and found to be 0.22 mg./I00 ml., using duplicate analysis of 30 samples. This compares well with the standard deviation for blind duplicates for the EDTA-calcium titration method for determining serum calcium, which was determined and found to be 0.27 mg./100 ml. using duplicate analyses of 40 samples. In contrast to the blind duplicate study, known duplicate data on 40 samples for serum calcium gave standard deviations of 0.15 mg./100 ml. for the AAS and 0.09 mg./100 ml. for the EDTA-calcium titration methods respectively. Comparison of the Serum Method with Oxalate Precipitation

The test method for serum, direct dilution of serum 1:50 in 0.1% lanthanum and determination of calcium content by atomic absorption Table 4.

CALCIUM

CONCENTRATION

3 DIFFERENT

AAS

IN 7 URINE

METHODS

(MG./i00

SPECIMENS

ML.;

1:50

AS

DETERMINED

BY

DILUTION)

Diluent Urine

No.

46-939 55-493 46-388 32-977

removed

by oxalate

precipitation,

ashed

La

0.1%

5.7 5.4 9.4 2.2 7.0 18.4 25.7

5.3 6.0 9.4 2.5 6.9 18.4 25.8

4.7 4.5 7.3 2.0 5.1 13J 18.0

46-243 46-238 55-574 *Ca

0.5%

Water

to Ca

carbonate,

and

diluted

in 0.1%

La.

La

TRUDEAU

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& FREIER

Clinical

Chemistry

spectrophotometry was compared to a method involving isolation of calcium by oxalate precipitation, ashing to carbonate, dilution 1 :50 in 0.1% lanthanum, and determination of calcium concentration by atomic absorption spectrophotometry. Thus, 2 atomic absorption methods 15

Fig. 3. Comparison of

direct

c

.2

10

dilution

in

seruni

for AAS analysis 40 determinations,

2

with

t

cmm with oxalate lowed by ashing

isolation

carbonate

0.1%

in

y, x; x2

4

=

r =

5

10 AAS

in

of

and

cal-

folto

solution

lanthanum.

-0.013 + 1.005 +0.96; Sy1 0.31; bias

-0.03; SD5 = t = 0.57; p>0.5.

0

of

lanthanum

0.33;

15

Precipitation X2

were compared: 1 in which the calcium was determined in the presence of serum and of all its potential interfering substances and 1 in which the calcium was removed from the serum. Figure 3 is a plot of the regression line for the 2 methods. The coefficient of correlation is +0.96, the standard error of the estimate-a measure of dispersion about the regression line in the y direction-is ±0.31 mg./100 ml. and the bias is small and cannot be shown to be significant by the t test. The standard deviation of the difference between the 2 methods for each specimen is ±0.33 mg./100 ml. Comparison of the Serum Method with EDTA-Calcium

Titration

The test method for serum was then compared to an entirely independent method, the chelatometric titration of calcein-calcium complex with EDTA (4). By inspection of Fig. 4 it may be seen that the agree-

CALCIUM

Vol. 13, No. 2, 1967

IN URINE AND

111

SERUM

ment is excellent. The correlation coefficient is +0.98 and the error of the estimate is ±0.25 mg./100 ml. A small bias was exist between the methods, significant at the 0.01’ level: on the the AAS serum method gave results 0.2 mg./100 ml. higher

standard found to average than the

15

Fig. 4.

Comparison

of direct dilution of serum in 0.1% lanthanum for AAS analysis (40 determinations) with direct titration by EDTA with calcein indicator. y1 = -0.37

+ +0.98; bias 0.42; 0.01.

1.064 x,; r = 5.-O25 = -0.21; SD,, = t = 3.02; p


I0

S

4

5

5

10

15

EDTA Colcein X

E]JTA calcium

method. The standard deviation for the differences by the 2 methods is ±0.42 mg./100 ml.

of serum

Comparison of the Urine Method with Oxalate Precipitation

Figure 5 is a regression line for results obtained for urine calcium by 2 methods performed on 40 consecutive urine samples. The test method consists of direct dilution of an acidified aliquot of urine 1 :50 in 0.5% lanthanum and determination by atomic absorption spectrophotometry, and the reference method consists of isolation of calcium by oxalate precipitation using the same acidified aliquot, ashing to carbonate, dilution 1:50 in 0.1% lanthanum, and determination by atomic absorption spectrophotometry. Here the correlation coefficient is 1.00 and the standard error of the estimate over a range of concentrations twice that of serum is ±0.71 mg./100 ml. There is a snlall bias that is not significant by the t test and the standard deviation of the difference of the methods is ±0.40 mg./100 ml.

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Discussion The use of lanthanum diluent eliminated not only the chemical binding interference of calcium absorption by phosphates and proteins, as may be seen from these experiments, but also avoided the ionizationsuppression effects of sodium. In the air-acetylene flame approxi60

50 Fig. 5.

Comparison

of direct dilution of urine in 0.5% lanthanum for AAS analysis (40 determinations) with isolation of cal-

>

g 2 30

cium with oxalate lowed by ashing

folto

carbonate and solution in lanthanum. y =

+

0.02

20

+1.00; bias 0.40; 0.6.

l0

10

20

30

40

50

0.994 S,+0.03;

=

t

x;

r

=

0.71; SD,, = 0.45; p> =

60

AAS Direct Dilution X

mately

10% of the calcium is lost from the ground state to the ionized state. If a metal that has a lower energy potential for ionization, i.e., sodium, is present in the flame in significant quantities, the calcium is more likely to remain in the ground state, and therefore have a higher absorption (14). Thus, it is seen that diluted serum samples containing sodium would have relatively more absorption when compared to solutions of calcium in water and relatively less when compared to solutions of calcium and sodium in water. However, in these experiments, when lanthanum in either 0.1% or 0.5% concentrations was used as the diluent in a 1 :50 dilution, differences in absorption could no longer be affected by adding sodium. In fact, recovery of calcium added to dilutions of calcium-free serums in 0.1% lanthanum was complete when compared to the absorption of standard calcium solutions iii the same lanthanum diluent.

Vol. 13, No. 2, 1967

CALCIUM

IN URINE AND

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113

The trichioracetic acid method of protein I)IeciI)itatioii prior to sample dilution was not studied as we did not find the presence of proteins in our prepared samples to be any particular problem with the 1:50 dilution. It was necessary to clean the burner only before each run, and rarely, if ever, during a run. The multistep method of protein precipitation by trichloracetic acid seems unduly complicated if acceptable results can be obtained by a simpler method-namely, direct dilution of the sample. The method of determining calcium in serum by direct dilution in 0.1% lanthanum and determination of calcium absorption by atomic absorption spectrophotometry agrees well with an entirely independent method, the EDTA-calcein complexometric method. The variation between methods is indeed small and corresponds to the standard deviation for double-blind duplicates by the atomic absorption method only. By isolating the calcium from the serum samples before determining calcium absorption-thus, completely avoiding the presence of serum proteins, phosphates, and sodium-results obtained were identical within the limits of variation to the results obtained when calcium absorption was determined in the presence of serum using 0.1% lanthanum diluent. The same was found to be true for urine; i.e., 0.5% lanthanum eliminates or avoids whatever substances there are in urine that may interfere with calcium absorption. Zettner and Seligson (10) suggested that the complexity of urine composition would detract from the accuracy of a direct dilution technic for urine calcium. The data in Figure 5 in which the calcium isolated from interferences by oxalate precipitation from 40 different urine specimens agree well with the direct dilution as well as the specific interferences evaluated in Table 2 support the view that a 1 :50 dilution of urine in 0.5% lanthanum is indeed suitable for urine calcium analysis. The simplicity and ease of the direct lanthanum dilution method for serum and urine calcium by atomic absorption spectrophotometry are also points in the method’s favor. By using any type of automatic or semiautomatic diluting device, specimens are prepared in seconds. Furthermore, it has been our experience using a digital read-out accessory for the Perkin-Elmer 303 that accurate results can be obtained directly in milligrams/100 ml. in approximately 1 or 2 minutes with no calculations necessary.

References 1. 2.

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. Citein. 63, 461 (1925). Sobel, A. E., and Sobel, B. A., The determination of calcium ill urine. J. Lab. Clin. Med. 26, 585 (1940).

114 3.

4.

5. 6. 7. 8. 9. 10. 11. 12. 13.

14.

TRUDEAU

& FREIER

Clinical

Chemistry

Homer, W. H., Determination of calcium in biological niaterial. J. Lab. Clin. Med. 45, 951 (1955). Bachra, B. N., Dauer, A., and Sobel, A. E., The complexometric titration of micro and ultramiero quantities of calcium in blood serum, urine and inorganic sait solutions. Clin. Chem. 4, 107 (1958). Appleton, H. D., West, M., Mandel, M., and Sala, A. M., The rapid determination of calcium in biologicalmaterial. Clin. Chem. 5, 36 (1959). Chen, P. S., and Toribara, T. Y., Determination of calcium in biological material by flame photometry. Anal. Chem. 25, 1642 (1953). Willis, J. B., The determination of metals in blood serum by atomic absorption spectroscopy. I. Calcium. Speotrochi’rm. Acta 16, 259 (1960). Walsh, A., The application of atomic absorption spectra to clinical analysis. Spectrochim. Acta 7, 108 (1955). Russell, B. J., Shelton, J. P., and Walsh, A., An atomic absorption spectrophotometer and its application to the analysis of solutions. Spectrochim. Acta 8, 317 (1957). Zettner, A., and Seligson, D., Application of atomic absorption spectrophotometry in the determination of calcium in serum. Clin. Chem. 10, 869 (1964). Sunderman, F. W., and Carroll, J. E., Measurement of serum calcium and magnesium by atomic absorption spectrophotometry. Am. J. Clin. Path. 43, 302 (1965). Operations Manual, Perkin-Elmer 303 Atomic absorption spectrophotometer. Instrument Division, Perkin-Elmer Corp., Norwalk, Conn. Treat, D. J., and Slavin, W., Clinical application of an atosnic absorption spectrophotometer linear in concentration. Atomic Absorption Newsletter 4, 300 (1965). Instrument Division, Perkin-Elmer Corp., Norwalk, Conn. Willis, J. B., Analysis of biological materials by atomic absorption spectrophotometry.

Methods

Biochem.

Anal.

11, 1 (1963).

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