IMPROVED METHOD FOR DETERMINATION OF CALCIUM AND MAGNESIUM IN BIOLOGIC

FLUIDS BY EDTA TITRATION

MARIE H. CARR, M.A., AND HUGH A. FRANK, M.D.

Research Laboratories, Veterans Administration Hospital, Kansas City, Missouri, and Department of Surgery, University of Kansas School of Medicine, Kansas City, Kansas

The increasing importance of studying the metabolism of calcium and mag­nesium has led to a demand in clinical chemistry for simple, rapid, and more accurate methods of analysis for these substances in biologic fluids. In response to this demand, a number of procedures based on a variety of principles have been proposed. One of the most promising of these is the titration of calcium and magnesium with a chelating agent. This paper deals with a procedure that was derived from the essential features of the reports of several earlier work­ers.i, 8,9, u, i2,2i-23,25 These have been redesigned into a system of analysis that is believed to be an improvement over any single one of its predecessors.

METHODS

Principle of analysis. Calcium in the specimen is separated from magnesium by precipitation as calcium oxalate. It is then washed, redissolved, and titrated with disodium ethylenediamine tetraacetate (EDTA), using eriochrome black T (EBT) as the indicator of the end point. Magnesium is titrated in an aliquot of the supernatant fluid, using the same reagents.

Reagents. Use freshly distilled or deionized water for preparation of the various reagents.

1. Stock standard for calcium (100 mEq. per 1.). Use reagent grade precipi­tated calcium carbonate, low in alkalies and chlorides, dried to constant weight at 110 C. Dissolve 2.502 Gm. in 5 to 10 ml. of concentrated hydrochloric acid, and dilute to 500 ml. This solution may be stored indefinitely in a polyethylene bottle.

2. Stock standard for magnesium (40 mEq. per 1.). Use reagent grade mag­nesium oxide that has been heated (at a red heat) in a platinum crucible until the weight is constant. Dissolve 0.4032 Gm. in 5 to 10 ml. of concentrated hydro­chloric acid, and dilute to 500 ml. Store the solution in a polyethylene bottle.

3. Mixed working standard (calcium, 5 mEq. per 1.; magnesium, 2 mEq. per 1.). Mix 5 ml. of each of the stock standard solutions, and dilute to 100 ml. with water.

4. Prepare a saturated solution of ammonium oxalate in water. 5. Solution of ammonium hydroxide. Dilute 2 ml. of concentrated solution of

ammonium hydroxide (28 per cent) to 100 ml. with water. (5. Solution of perchloric acid. Dilute 20 ml. of reagent grade perchloric acid

(70 per cent) to 100 ml. with water.

Received, April 5, 1956; accepted for publication June 4. Mrs. Carr is Chief Biochemist, and Dr. Frank is Director of the Research Laboratories.

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1158 CAHR AND FRANK Vol. 26

7. Solution of ethanolamine. Dilute 10 ml. of reagent grade (or redistilled) ethanolamine to 100 ml. with water. Impure ethanolamine is not satisfactory, inasmuch as the end point of the titration would not be reliable. The solution of ethanolamine should be freshly prepared every 2 or 3 days.

8. Magnesium-EDTA complex. Dissolve 2 Gm. of magnesium chloride in approximately 25 ml. of water. Dissolve 7.5 Gm. of EDTA in approximately 75 ml. of water, add the solution to the magnesium chloride, and mix. When precipi­tation is complete, filter the mixture, collect the Na2Mg complex, wash it repeat­edly with small quantities of cold distilled water, and then dry it at room tem­perature.

9. Stock solution of EDTA. Dissolve 4.5 Gm. of reagent grade EDTA in water and dilute the solution to 1 1. This solution may be stored indefinitely in a polyethylene bottle.

10. Working solution of EDTA for titration of calcium. Dilute 50 ml. of the stock solution to 500 ml., and store in a polyethylene bottle.

11. Working solution of EDTA for titration of magnesium. Dilute 25 ml. of the stock solution to 500 ml., and store in a polyethylene bottle.

12. Solution of EBT. Dissolve 0.1 Gm. of EBT in 25 ml. of methanol, and add 1 ml. of concentrated solution of ammonium hydroxide. This solution will be stable for approximately 2 weeks.

13. Solution of bromcresol green. Grind 0.1 Gm. of bromcresol green in a mor­tar with 2.9 ml. of 0.05 N sodium hydroxide, and dilute the solution to 400 ml. with water.

Glassware. Calcium is precipitated from the samples, washed, redissolved, and titrated in graduated centrifuge tubes. This procedure limits the volume of the titrant; thus, a more concentrated solution of EDTA is used for the titration of calcium. The solution is delivered from a 10-ml. buret that is graduated in 0.05-ml. markings.

Magnesium is titrated in 25-ml. Erlenmeyer flasks, using the same type of buret. A more dilute solution of EDTA is used in order that the volume of the titrated mixture will be large enough for sufficient accuracy of the results.

Procedure. One-ml. samples are used routinely, but 0.5-ml. samples will suffice. The mixed standard is routinely analyzed in duplicate (with the unknown samples) as a check on the manipulations and the reagents, as well as a compar­ative basis for the color of the end point. If a particular unknown sample contains an unusually high level of calcium or magnesium, it is advisable to dilute the specimen prior to analysis, in order to avoid undue dilution of the buffer by the titrant. Specimens of urine should be acidified with hydrochloric acid in order to redissolve any salts of calcium that may have precipitated during the period prior to removal of the samples to be analyzed.

1. To each tube containing 1.0 ml. of the mixed standard solution, or 1.0 ml. of the sample to be analyzed, add 2.5 ml. of distilled water and then 0.5 ml. of saturated solution of ammonium oxalate. Samples of urine or gastric secretion may require adjustment of the pH at this time; add 1 drop of the dilute solution of bromcresol green, and adjust the pH with ammonia or acetic acid so that the sample has a green tint. Mix the contents well by tapping the tubes, and set

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Oct. .1956 CALCIUM AND MAGNESIUM DETERMINATION 1.159

them aside for not less than 2 hours, in order to provide time for complete precipitation.

2. Centrifuge the tubes for 15 minutes at approximately 900 g. Decant the supernatant fluid, and set it aside for the analysis of magnesium. Invert the tubes on filter paper for approximately 5 minutes.

3. By means of a wash-bottle with a fine nozzle, break up the precipitated calcium oxalate and rinse down the side of the tube, using a total of approximately 5 ml. of the dilute solution of ammonium hydroxide.

4. Centrifuge for 10 to 15 minutes (900 </), decant, and discard the supernatant fluid. Loosen the precipitate by tapping the tubes.

5. Dissolve the precipitate with 1 ml. of the solution of perchloric acid. Add 3 ml. of ethanolamine-magnesium-EDTA complex (approximately 10 mg. of magnesium-EDTA complex per 100 ml. of the solution of ethanolamine). Add 2 or 3 drops of the solution of EBT, and titrate immediately with the working solution of EDTA for analysis of calcium. If the tubes are allowed to stand for more than 5 minutes after the addition of the ethanolamine buffer, samples with high levels of calcium may undergo a reprecipitation of the calcium oxalate at the relatively high pH that is present (approximately 10).

6. The end point of the titration is a clear blue with a slight tinge of green. I t is not so important that the color be exact, as it is that an identical color be reproduced in each tube, and this is regularly attained after a little practice. The color of the end point is not stable, but gradually fades on standing, and this precludes the use of a single analysis of the standard solution as a color reference during a series of titrations.

7. Calculation of the level of calcium.

titration of the sample , . . ,. . , , , . . . . , X calcium m the standard = calcium in the sample titration of the standard

8. To a 3.0-ml. aliquot of the supernatant fluid collected after precipitating calcium from the sample, add 3 ml. of the solution of ethanolamine and 2 or 3 drops of the solution of EBT.

9. Titrate immediately with the working solution of EDTA for the analysis of magnesium. The end point is similar to that of the titration for calcium, with the exception that the yellow pigment in serum tends to increase the tinge of green. The titration should be performed slowly, especially near the end point.

10. Calculation of the level of magnesium is done in the same manner as that, for calcium, using the proper value for magnesium in the standard.

Other methods. Several previously published technics were included in this study, in order to provide a means of comparing results of the analyses. The following methods were performed according to the published directions, except that the method of Sobel and Hanok was adapted to a macro procedure:

1. Titration with permanganate (Kramer and Tisdall),17 as modified by Clark and Collip4 and by Sendroy.24 :

2. Photometric (alizarin complex) method of Natelson, Penniall, and Secard.18'19

3. Flame photometric methods of Kingsley and Schaffert,16-I6 and of Kapus-

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1160 CARR AND FRANK Vol. 26

cinski, Moss, Zak, and Boyle,14 for the determination of calcium and magnesium. as well as that of Davis5 for the analysis of magnesium.

4. Titration with EDTA, as proposed by Holasek and Flaschka,12 Buckley, Gibson, and Bartolotti,2 Sobel and Hanok,25 Friedman,8 Friedman and Rubin,9

and Greenblatt and Hartman.10

5. Photometric (titan yellow) method for analysis of magnesium, as proposed by Orange and Rhein.20

RESULTS

The method described in this paper was developed in an attempt to improve upon previously published technics that are listed in the preceding section. The proposed method has been used (as described) for almost 2 years, and hundreds of samples were satisfactorily analyzed for their content of calcium and mag­nesium. The validity of the method was evaluated in different ways.

On a number of occasions, aliquots of the same specimen were analyzed by 2 or more methods in order to compare the results. Data from 1 study of this sort are listed in Table 1, representing analyses of a single pool of human serums by means of 6 different methods. Each of the methods was used to analyze 3 types of samples from the pool of serums, i.e., an unaltered sample, a diluted sample, and a sample to which a known amount of calcium was added. In all instances, 2 or more replicates were analyzed. Such data not only indicate systematic differences among the results of various procedures, but also (1) the reproducibility of results, (2) the effect of altering the total chemical pattern by dilution, and (3) the accuracy of each method in the recovery of calcium that is added to a sample.

TABLE 1

COMPARISON OF 6 M E T H O D S FOR D E T E R M I N I N G CALCIUM IN SERUM

Method*

1 2 3 4 5 6

Undiluted Serum

Meant value

5.39 5.26 4.80 4.80 4.70 4.82

Standard deviation

0.05 0.02 0.03 0.07 0.07 0.03

Serum Diluted 2:3

Meant value

3.59 3.31 3.OS 3.60 3.10 3.22

Standard deviation

0.04 0.01 0.06 0.40 0.07 0.03

Theo­retical content

3.59 3.50 3.20 3.20 3.10 3.21

Error

0 - 0 . 1 9 - 0 . 1 2 + 0 . 4 0

0 + 0 . 0 1

Serum with Added Calcium (2 mEq./l.)

Meant value

7.28 7.06 7.32 7.00 6.90 6.78

Standard deviation

0.04 0 0

0.20 0.07 0.08

Theo­retical

content

7.39 7.26 6.80 6.80 6.70 6.82

Error

+0 .01 - 0 . 2 0 + 0 . 5 2 + 0 . 2 0 +0 .20 - 0 . 0 4

* 1. Ti t ra t ion with E D T A (murexide indicator) (Greenblatt and Har tman) . 1 0

2. Flame photometry method proposed by Kingsley and SchalTert.16

3. Ti t ra t ion with potassium permanganate , as modified4' " from the method of Kramer and T i sda l l . "

4. Determinat ion as alizarin complex (Natelson, Penniall , and Secard).1 9

5. T i t ra t ion with EDTA (EBT indicator) , as modified from the method of Sobel and H a n o k . "

6. Ti t ra t ion with E D T A (EBT indicator) , as described in this paper. t Values are listed in mEq. per 1., and represent the means calculated from triplicate

determinat ions.

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TABLE 2

COMPARISON OF M E T H O D S FOB D E T E R M I N I N G CALCIUM AND MAGNESIUM IN BIOLOGIC

F L U I D S OTHER T H A N SERUM

Specimen*

Spinal fluid Urine Gastric secretion Bile, ashed and re-

dissolved

Determination of Calcium

Titration with potassi­um permanganate

Mean value*

mEq./l.

2.44 3.93 1.51 2.87

Standard deviation

mEq./l.

0.03 0.06 0.05 0.02

Titration with EDTAf

Mean value*

mEq./l.

2.36 3.89 1.36 2.91

Standard deviation

mEq./l.

0.02 0.04 0.04 0.04

Determination

Titration with KDTAf

Mean value*

mEq./l.

2.27 5.97 0.69 2.04

Standard deviation

mEq./l.

0.04 0.08 0.05 0.06

of Magnesium

Titan yellow method

Mean value*

mEq./l.

2.5 6.1 0.6 2.0

Standard deviation

mEq./l.

0.0 0.5

0 0

* Values arc the means calculated from determinations on 3 aliquots of the same speci­men of each fluid.

f Method described in this paper.

TABLE 3

REPRODUCIBILITY OF R E S U L T S OF ANALYSES ON A STANDARD P O O L OF S E R U M *

Specimen

Pool of human serum (20

Mean Standard deviation Actual range of values 2 Sigma range of values Maximum error

analyses)

Determination of Calcium

mEq./l.

4.62 4.75 4.64 4.59 4.66 4.57 4.50 4.48 4.52 4.60 4.71 4.55 4.62 4.57 4.62 4.34 4.57 4.63 4.5S 4.63

4.59 0.09

4.34 to 4.75 4.50 to 4.6S

0.25

Determination of Magnesium

mEq./l.

1.77 1.75 1.65 1.96 1.76 1.6S 1.74 1.60 1.80 1.64 1.72 1.70 1.60 1.74 1.70 1.79 1.79 1.68 1.69 1.69

1.72 O.OS

1.60 to 1.96 1.64 to 1.S0

0.24

* The individual values represent the results of repeated analyses of a single pool of human serum. These were performed by 3 analysts a t the same time tha t analyses on routine specimens were being performed, using the method described in this paper.

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1162 CARR AND FRANK Vol. 26

TABLE 4 RECOVERY OF CALCIUM AND MAGNESIUM ADDED TO SERUM AND URINE*

Specimen

Serum Undiluted Diluted 1:2 with water

Urine Undiluted Diluted 1:2 with water

Calcium Added

mEq./l.

0 0

2.50 2.00 1.50 1.00 0.50

0 0

2.50 2.00 1.50 1.00 0.50

Calcium Foundt

mEq./l.

4.59 2.32 4.80 4.42 3.84 3.32 2.86

3.90 1.95 4.49 4.05 3.38 2,96 2.46

Theo­retical

Content of Calcium

mEq./l.

2.30 4.80 4.30 3.80 3.30 2.80

1.95 4.45 3.95 3.45 2.95 2.45

Error}

mEq./l.

- 0 . 0 2 0

+ 0 . 1 2 +0 .04 +0 .02 +0 .06

0 +0 .04 +0 .10 - 0 . 0 7 + 0 . 0 1 +0 .01

Mag­nesium Added

mEq./l.

0 0

0.50 1.00 1.50 2.00 2.50

0 0

0.50 1.00 1.50 2.00 2.50

Mag­nesium Foundf

mEq./l.

1.71

0.90 1.35 1.85 2.25 2.81 3.36

3.63 1.81 2.42 2.83 3.36 3.84 4.16

Theo­retical

Content of Magnesium

mEq./l.

0.S6 1.36 1.86 2.36 2.86 3.36

1.S2 2.32 2.S2 3.32 3.82 4.32

Errort

mEq./l.

+ 0 . 0 4 - 0 . 0 1 - 0 . 0 1 - 0 . 1 1 - 0 . 0 4

0

- 0 . 0 1 + 0 . 1 0 +0 .01 +0 .04 + 0 . 0 2 - 0 . 1 6

* Calcium and magnesium were added to the same aliquots of sample in each instance, but in reciprocally varying amounts. Thus, the validity of the separation of these ions was tested, as well as the recovery of each.

f Each value is the mean of 4 analyses of serum, and 2 of urine. % Standard error for determination of calcium in serum = 0.06 mEq. per 1.

Standard error for determination of magnesium in serum = 0.06 mEq. per 1. Standard error for determination of calcium in urine = 0.06 mEq. per 1. Standard error for determination of magnesium in urine = 0.09 mEq. per 1.

The direct titration of calcium with EDTA (using murexide as an indicator) and the flame photometric method, as they were performed in this study, yielded results that were consistently higher than those obtained by other procedures. On the other hand, the precision of these 2 methods (as indicated by the variation between replicates) and their accuracy (as indicated by recoveries of added calcium) are within acceptable limits. These 2 methods consistently yield values for serum calcium that are significantly higher than those obtained by titration with permanganate or by EDTA-titration of calcium isolated from the sample. This was true not only of the results obtained in this illustrative study, but whenever such data were compared.

Analyses of magnesium by the presently proposed method were compared only with the photometric (titan yellow) technic suggested by Orange and Rhein.20 In a series of 50 consecutive specimens of normal and abnormal human serum, analyzed by the 2 methods, the difference between the values (for a single specimen) ranged from +0.4 mEq. per 1. to —0.2 mEq. per 1. The mean of the differences between the results of the 2 methods (i.e., titan yellow and titration with EDTA) was +0.05 mEq. per 1. In order to verify the premise that

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Oct. 1956 CALCIUM AND MAGNESIUM DETERMINATION 1163

calcium is quantitatively precipitated from serum (by treating with ammonium oxalate, under the conditions of the proposed method), a series of duplicate samples were analyzed directly, and then after ashing in platinum crucibles. The results were in excellent agreement.

Spinal fluid, urine, fluid obtained by aspiration of the stomach, and bile are complex mixtures of greatly different composition, and a method that is suitable for the analysis of serum is not necessarily satisfactory for analysis of these other biologic fluids. Therefore, the proposed procedure was further evaluated by comparing results with those obtained by other methods for the analysis of such fluids. The data derived from 1 such study are listed in Table 2.

A large pool of human serum was collected during July 1955. It was thoroughly

TABLE 5

RECOVERY OK CALCIUM AND MAGNESIUM ADDED TO SPINAL F L U I D , GASTRIC ASPIRATION

F L U I D , AND B I L E

Spinal fluid: Calcium:

Found* Theoretical content. Difference

Magnesium: Found* Theoretical content Difference

Gastric Aspiration Fluid: Calcium:

Foundf Theoretical content Difference

Magnesium: Foundj Theoretical content Difference

Bile (ashed): Calcium:

Foundf Theoretical content Difference

Magnesium: Foundf Theoretical content. Difference

Undiluted Fluid

0 0

2.39

2.31

1.38

0.68

2.91

2.04

mEq./l. 2.00 1.00

4.55 4.39

+0 .16

3.27 3.31

- 0 . 0 4

3.43 3.38

+ 0 . 0 5

1.71 1.6S

+0 .03

5.04 4.91

+ 0 . 1 3

3.06 3.04

+0 .02

1.00 2.00

3.42 3.39

+ 0 . 0 3

4.22 4.31

- 0 . 0 9

2.36 2.3S

- 0 . 0 2

2.77 2.68

+0 .09

3.87 3.91

- 0 . 0 4

3.86 4.04

- 0 . 1 S

Fluid Diluted 1:2

0 0

L I S 1.20

- 0 . 0 2

1.40 1.16

- 0 . 2 4

0.67 0.69

- 0 . 0 2

0.46 0.34

+0 .12

1.43 1.46

- 0 . 0 3

1.05 1.02

+0 .03

mEq./l. 2.00 1.00

3.23 3.20

+ 0 . 0 3

2.21 2.16

+ 0 . 0 5

2.56 2.69

- 0 . 1 3

1.42 1.34

+0 .08

3.54 3.46

+0 .0S

2.02 2.oi 0.00

1.00 2.00

2.13 2.20

- 0 . 0 7

3.11 3.16

- 0 . 0 4

1.47 1.69

- 0 . 2 2

2.37 2.34

+0 .03

2.52 2.46

+0 .06

2.99 3.02

- 0 . 0 3

Fluid Diluted 2:3

(1 0

1.54 1.59

- 0 . 0 5

1.61 1.54

+0 .07

O.SS 0.92

- 0 . 0 4

0.59 0.45

+0.14

1.9S 1.94

+ 0 . 0 2

1.44 1.36

+ 0 . 0 8

mEq./l. 2.00 1.00

3.57 3.59

- 0 . 0 2

2.53 2.54

- 0 . 0 1

2.97 2.92

+ 0 . 0 5

1.55 1.45

+0 .10

4.10 3.94

+0 .16

2.41 2.36

+ 0 . 0 5

1.00 2.00

2.53 2.59

- 0 . 0 6

3.51 3.54

- 0 . 0 3

1.79 1.92

- 0 . 1 3

2.45 2.45 0

2.94 2.94 0

3.34 3.36

- 0 . 0 2

* Mean value for 2 determinations, f Mean value for 3 determinations.

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1.1 04 CAR It AND FRANK Vol. 26

mixed, divided into small portions, and sealed in ampuls prior to storage in a deep-freeze. From time to time during a period of 8 months, portions of this pool of serum were analyzed concomitantly with routine samples, the procedures being performed by 3 analysts in this laboratory. Such a plan served not only as a means of evaluating the reproducibility of results by the proposed method, but also as a batch-check on the accuracy of the routine analyses. Table 3 contains the data collected from this standard pool of serum. The standard deviations resulting from analyses by the proposed procedure were 2.0 per cent of the mean value for serum calcium, and 4.6 per cent of the mean value for serum magnesium.

As a further check on accuracy of results, dilution and recovery experiments were performed on all of the various biologic fluids for which the method was adapted. These were designed to demonstrate the validity of the separation of calcium from magnesium (by precipitating calcium as the oxalate salt) under the conditions of the proposed method, as well as the recovery of added amounts of calcium and magnesium. Data from some of these studies are compiled in Tables 4 and 5.

DISCUSSION

Analysis of calcium by means of precipitation as calcium oxalate, and quantita­tion of this salt by titration with permanganate, has withstood the test of time, and this method has become a standard, widely used procedure. Newly proposed methods should be evaluated in relation to such a technic, but it has certain disadvantages for routine use. Dilute solutions of permanganate are relatively unstable, the titration must be performed with a hot sample, traces of organic material may also be oxidized by permanganate, and the reagent is not suitable for the analysis of magnesium. Although flame photometry greatly simplifies the determination of sodium and potassium, the results with analyses of calcium and magnesium are disappointing. Inasmuch as the determinations are greatly influenced by the total chemical pattern of the sample, flame photometry may not be used for biologic fluids other than serum unless calcium and magnesium are first separated from the sample. Furthermore, even with serum, the procedure yields results that tend to be higher than those obtained with other methods. The photometric method with alizarin is accurate, but careful control of the procedure for extraction is required, and the reagents have a disagreeable odor. The procedure with titan yellow for the determination of magnesium is a simple photometric method, but, in our experience, it is less precise than the titrimetric method with EDTA. For these reasons, the titration of calcium and magnesium with EDTA seems to be the best method for use with a variety of biologic fluids.

EDTA methods. Schwarzenbach intensively studied chelating agents in recent years, and analytic methods for the determination of calcium and magnesium were developed as a result of his work and that of others. These were applied first to the analysis of water, and later to biologic fluids. We have utilized this work, especially the papers and monograph of Schwarzenbach1 •22 •23 and those of Holasek and Flaschka,12 Sobel and Hanok,25 and Friedman,89 in developing the proposed present system of analysis.

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Od. 1956 CALCIUM AND MAGNESIUM DETERMINATION 1165

Most of the methods previously published depend upon using EDTA for the direct titration of the sum of divalent cations in the sample, with eriochrome black T as the indicator of the end point. From this point on they differ. The method of Buckley and his associates2 is an example of a technic where calcium is titrated directly with murexide as the indicator, and the level of magnesium is calculated by difference. Another approach (used by Sobel and Hanok)25 is to determine the amount'of magnesium by means of titan yellow (or assume that it is present in a normal, unvarying amount), and then calculate the level of calcium by the difference. The method of Friedman8'9 represents a third type of procedure, i.e., precipitate the calcium and titrate the magnesium in the super­natant; fluid with EDTA and eriochrome black T, and then calculate the content of calcium by difference. There are several variations of technic reported for each of these 3 major approaches, including suggestions for photometric titra­tion6'7 and for back-titration10'20 from an excess of EDTA.

The procedure proposed in this paper represents still another technic, i.e., the direct titration of calcium and magnesium after separating them by pre­cipitation with ammonium oxalate. This method is somewhat similar to that of Holasek and Flaschka,12 who believed that the presence of blood protein made it impossible to titrate magnesium. In order to solve this problem, they prepared a protein-free filtrate by precipitating serum with trichloracetic acid. They recognized that such filtrates yielded higher values than those obtained with serum or serum ash. Such differences are apparently the result of volume dis­placement, as discussed by Sendroy.24 In the present study it was found that the ethanolamine buffer (instead of ammonia-ammonium chloride) allowed satisfac­tory titrations of magnesium with the protein present. Early in the work, it was noted that the sum of the separate titrations of calcium and of magnesium did not regularly correspond to the direct titration of the sum of divalent cations in the serum. This is believed to result from binding of calcium by proteins, rather than binding of magnesium. The preparation of a filtrate with trichloracetic acid seems to be unnecessary, as well as undesirable.

Several aspects of the proposed procedure are deserving of emphasis or com­ment:

1. The glassware should be pyrex, and it should be carefully rinsed. All of the reagents and dilutions must be made with freshly distilled or deionized water that is stored in polyethylene containers. If these precautions are not used, small amounts of contaminating calcium and magnesium will produce detectable errors.

2. The presence of ferric or ferrous iron as a contaminant, or as a constituent of the samples to be analyzed, makes the detection of the end point difficult, or even impossible, when titrating magnesium. Attempts to solve the problem of dealing with iron in certain samples of bile and fluid obtained by aspirating the stomach have been unsuccessful.

3. Small amounts of hemolysis in the samples of serum do not interfere with the analyses.

4. The precipitation of calcium oxalate in this procedure is subject to the same criticisms24 as it is in any other such method, inasmuch as it is empirical

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.1.166 CARK AN! I) FRANK Vol. 26

and dependent on the conditions that are present, especially the pl l . As pre­viously suggested by Sendroy24 and Holth,13 we found that treatment with a saturated solution of ammonium oxalate at a pH near 5 will reliably separate calcium from magnesium (Tables 4. and 5). Less concentrated solutions of ox­alate, as suggested by others,8, '• 12 were not satisfactory in our experience with this technic. We also found that at least 2 hours, and preferably 4 hours, should be allotted for complete precipitation of the calcium.

5. If the patient has been treated with intravenous injections of EDTA in order to augment the excretion of calcium, the pH of the samples must be less than 5* or calcium will not quantitatively separate from the EDTA and be precipitated as the oxalate salt.3 Samples from such patients will already contain EDTA in the supernatant fluid (with magnesium), and, therefore, the samples are not suitable for titrations of magnesium.

6. The ethanolamine buffer suggested by Sobel and Hanok25 was preferable to the ammonia-ammonium chloride buffer proposed by Holasek and Flaschka12

and by Friedman,8,9 inasmuch as magnesium (in the supernatant fluid) may be satisfactorily titrated only when the former buffer is used. On the other hand, either of these buffers may be used for the titration of calcium.

7. Biedermann and Schwarzenbach1 were the first to indicate that the end point ultimately depends on the titration of magnesium when EBT is used as the indicator. Thus, it is necessary to add magnesium-EDTA complex to the isolated calcium in order to develop the red color from which titration to blue is performed.

SUMMARY

The authors describe a method for the analysis of calcium and magnesium in serum and other biologic fluids. The new procedure represents a modification of previously published methods, and it is based on (1) the separation of calcium from magnesium by quantitative precipitation of calcium oxalate, and (2) the titration of each of the ions separately with ethylenediamine tetraacetate (EDTA), using eriochrome black T (EBT) as the indicator of the end point. The proposed method was critically evaluated by means of experiments to test reproducibility and recovery, and also by comparison of its results with those of other methods. The new procedure has advantages over the others in that it is simple and easy to perform, it yields results with greater accuracy, and it is suitable for routine use in clinical laboratories, as well as for inves­tigative studies.

SUMMAKLO IN liVT15RLI.NTC.UA

Le autores describe un methodo pro le analyse de calcium e magnesium in sero e altere fluidos biologic. Le nove inethodo representa un modification de methodos previemente publicate. Illo es basate super (!) le separation de calcium ab mag-

* After the addition of the saturated solution of ammonium oxalate, add a drop of solu­tion of bromcrcsol green, and adjust the reaction with acetic acid until the mixture has a green tint.

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Oct. 1956 CALCIUM AND MAGNESIUM DETERMINATION 1107

nesium per le precipitation quantitative de oxalato de calcium e (2) le titration separate de ille iones per medio de ethylenediamina-tetraacetato (EDTA), con nigro eriochrome T como indicator del puncto terminal. Le methodo hie proponite esseva evalutate per medio de experinientos destinate a probar su reproducibilitate e grado de rendimento e etiam per medio de comparationes de su resultatos con le resultatos obtenite per altere methodos. Le nove methodo ha le avantage, in comparation con alteres, que illo es simple e facile a executar, que illo produce resultatos de plus alte grados de exactitude, e que illo se presta al uso routinari in laboratories clinic e etiam al uso in studios investigatori.

Acknowledgment. The authors wish to express their appreciation for the able technical assistance of Miss Bet ty Huffman, Miss Mary Lou Brosnahan, and Mrs. Marjorie Wight, and for the assistance of Mrs. Peggy Brunkhorst in the preparation of the manuscript .

R E F E R E N C E S

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2. BUCKLEY, E. S., Jn . , GIBSON, J . G., I I , AND BARTOLOTTI, T. R . : Simplified t i t r imetric technique for assay of calcium and magnesium in plasma. J . Lab. & Clin. Med., 38: 751-761, 1951.

3. CARR, M. H., AND FRANK, H, A.: Unpublished da ta from this laboratory. 4. CLARK, E. P. , AND C O L L I P , J. B . : A study of the Tisdall method for the determination

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18. NATELSON, S., AND P E N N I A L L , R. : Colorimetric estimation of ultramicro quanti t ies of calcium in human scrum as the complex with alizarin. Anal. Chem., 27: 434-437,1955.

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22. SCHWARZENBACH, G.: Chelate complex formation as a basis for t i trat ion process. X I I International Congress of Pure & Applied Chemistry, Sept. 1951.

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