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.
1157
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 Na 2 Mg 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 0.05ml. 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
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.
It 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-
1160
CARR AND
Vol. 26
FRANK
cinski, Moss, Zak, and Boyle,14 for the determination of calcium and magnesium.
as well as that of Davis 5 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 I N SERUM
Undiluted Serum
Method*
1
2
3
4
5
6
Serum Diluted 2:3
Serum with Added Calcium (2 mEq./l.)
Meant
value
Standard
deviation
Meant
value
Standard
deviation
Theoretical
content
Error
Meant
value
Standard
deviation
Theoretical
content
Error
5.39
5.26
4.80
4.80
4.70
4.82
0.05
0.02
0.03
0.07
0.07
0.03
3.59
3.31
3.OS
3.60
3.10
3.22
0.04
0.01
0.06
0.40
0.07
0.03
3.59
3.50
3.20
3.20
3.10
3.21
0
-0.19
-0.12
+0.40
0
+0.01
7.28
7.06
7.32
7.00
6.90
6.78
0.04
0
0
0.20
0.07
0.08
7.39
7.26
6.80
6.80
6.70
6.82
+0.01
-0.20
+0.52
+0.20
+0.20
-0.04
* 1. T i t r a t i o n with E D T A (murexide indicator) (Greenblatt and H a r t m a n ) . 1 0
2. Flame p h o t o m e t r y method proposed by Kingsley and SchalTert. 16
3. T i t r a t i o n with potassium permanganate, as modified 4 ' " from the method of K r a m e r
and T i s d a l l . "
4. D e t e r m i n a t i o n as alizarin complex (Natelson, Penniall, and Secard). 1 9
5. T i t r a t i o n with E D T A (EBT indicator), as modified from the method of Sobel and
Hanok."
6. T i t r a t i o n with E D T A (EBT indicator), as described in this paper.
t Values are listed in m E q . per 1., and represent the means calculated from triplicate
determinations.
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 M A G N E S I U M
FLUIDS OTHER THAN SERUM
Determination of Calcium
Titration with potassium permanganate
Specimen*
Spinal fluid
Urine
Gastric secretion
Bile, ashed and redissolved
IN
BIOLOGIC
Determination of Magnesium
Titration with
EDTAf
Titration with
KDTAf
Titan yellow
method
Mean
value*
Standard
deviation
Mean
value*
Standard
deviation
Mean
value*
Standard
deviation
Mean
value*
Standard
deviation
mEq./l.
mEq./l.
mEq./l.
mEq./l.
mEq./l.
mEq./l.
mEq./l.
mEq./l.
0.03
0.06
0.05
0.02
2.36
3.89
1.36
2.91
0.02
0.04
0.04
0.04
2.27
5.97
0.69
2.04
0.04
0.08
0.05
0.06
2.5
6.1
0.6
2.0
2.44
3.93
1.51
2.87
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 O F R E S U L T S O F ANALYSES ON A STANDARD P O O L O F S E R U M *
Specimen
Determination of Calcium
Determination of Magnesium
mEq./l.
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
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
4.59
0.09
4.34 to 4.75
4.50 to 4.6S
0.25
1.72
O.OS
1.60 to 1.96
1.64 to 1.S0
0.24
Pool of human serum (20 analyses)
Mean
Standard deviation
Actual range of values
2 Sigma range of values
Maximum error
* T h e 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 t h a t analyses on
routine specimens were being performed, using the method described in this paper.
1161
1162
Vol. 26
CARR AND FRANK
TABLE 4
RECOVERY OF CALCIUM AND MAGNESIUM ADDED TO SERUM AND URINE*
Specimen
Serum
Undiluted
D i l u t e d 1:2 with water
Urine
Undiluted
Diluted 1:2 with water
TheoCalcium Calcium retical
Added Foundt Content of
Calcium
Error}
TheoMagMagretical
nesium nesium Content
of
Added Foundf Magnesium
Errort
mEq./l. mEq./l.
mEq./l.
mEq./l.
mEq./l. mEq./l.
mEq./l.
mEq./l.
0
0
2.50
2.00
1.50
1.00
0.50
4.59
2.32
4.80
4.42
3.84
3.32
2.86
2.30
4.80
4.30
3.80
3.30
2.80
-0.02
0
+0.12
+0.04
+0.02
+0.06
0
0
0.50
1.00
1.50
2.00
2.50
0.90
1.35
1.85
2.25
2.81
3.36
0.S6
1.36
1.86
2.36
2.86
3.36
+0.04
-0.01
-0.01
-0.11
-0.04
0
0
0
2.50
2.00
1.50
1.00
0.50
3.90
1.95
4.49
4.05
3.38
2,96
2.46
1.95
4.45
3.95
3.45
2.95
2.45
0
+0.04
+0.10
-0.07
+0.01
+0.01
0
0
0.50
1.00
1.50
2.00
2.50
3.63
1.81
2.42
2.83
3.36
3.84
4.16
1.S2
2.32
2.S2
3.32
3.82
4.32
-0.01
+0.10
+0.01
+0.04
+0.02
-0.16
1.71
* 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
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 M A G N E S I U M ADDED TO S P I N A L F L U I D , G A S T R I C
FLUID,
AND
Undiluted Fluid
0
0
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
mEq./l.
2.00
1.00
1.00
2.00
ASPIRATION
BILE
Fluid Diluted 1:2
0
0
mEq./l.
2.00
1.00
1.00
2.00
Fluid Diluted 2:3
(1
0
mEq./l.
2.00
1.00
1.00
2.00
2.39
4.55 3.42 L I S 3.23 2.13 1.54 3.57 2.53
4.39 3.39 1.20 3.20 2.20
1.59 3.59 2.59
+0.16 +0.03 - 0 . 0 2 +0.03 - 0 . 0 7 - 0 . 0 5 - 0 . 0 2 - 0 . 0 6
2.31
3.27 4.22 1.40 2.21 3.11 1.61 2.53 3.51
3.31 4.31 1.16 2.16 3.16 1.54 2.54 3.54
- 0 . 0 4 - 0 . 0 9 -0.24 +0.05 - 0 . 0 4 +0.07 - 0 . 0 1 - 0 . 0 3
1.38
3.43 2.36 0.67 2.56 1.47 O.SS 2.97 1.79
3.38 2.3S 0.69 2.69 1.69 0.92 2.92 1.92
+0.05 -0.02 -0.02 -0.13 -0.22 -0.04 +0.05 -0.13
0.68
1.71 2.77 0.46 1.42 2.37 0.59 1.55
1.6S 2.68 0.34 1.34 2.34 0.45 1.45
+0.03 +0.09 +0.12 +0.08 +0.03 +0.14 +0.10
2.45
2.45
0
2.91
5.04 3.87 1.43 3.54 2.52 1.9S 4.10
4.91 3.91 1.46 3.46 2.46 1.94 3.94
+0.13 -0.04 - 0 . 0 3 +0.0S +0.06 +0.02 +0.16
2.94
2.94
0
2.04
3.06 3.86 1.05
3.04 4.04 1.02
+0.02 -0.1S +0.03
* Mean value for 2 determinations,
f Mean value for 3 determinations.
2.02
2.99 1.44 2.41 3.34
3.02 1.36 2.36 3.36
0.00 - 0 . 0 3 + 0 . 0 8 + 0 . 0 5 - 0 . 0 2
2.oi
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, 8 9 in developing
the proposed present system of analysis.
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 Friedman 8 ' 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 titration 6 ' 7 and for back-titration 10 ' 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
.1.166
CARK AN! I) FRANK
Vol. 26
and dependent on the conditions that are present, especially the pll. 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 I N 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.
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. T h e authors wish to express their appreciation for t h e able technical
assistance of Miss B e t t y Huffman, Miss Mary Lou Brosnahan, and M r s . Marjorie Wight,
and for the assistance of Mrs. Peggy Brunkhorst in the preparation of the manuscript.
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