The Coleman flame photometer was
not satisfactory for urinary calcium de-
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concentrations of bilirubin and greater
hemolysis blanks could not be used
Clin. Med. 31, 1262-6 (1946).
(4) Ferro, P. V., Ham, A. B., Am. J.
terminations.
Effect of Hemolysis and Bilirubin.
Slight hemolysis and bilirubin con-
centrations as high as 4 mg. % had
no effect on the rate or completion of
color development of the NFR-
calcium color complex. At higher
Determination of Calcium and Magnesium in
Urine by Atomic Absorption Spectroscopy
J. B. WILLIS
Division of Chemical Physics, C.S.I.R. O. Chemical Research Laboratories, Melbourne, Australia
The calcium content of urine may
be determined by atomic absorption
measurement of specimens diluted 5-
to 50-fold with a solution of either
lanthanum chloride or strontium chloride
containing 1% by weight of the metal.
The solution is sprayed into an air-
acetylene flame. The values obtained
agree well with those obtained by the
oxalate permanganate
-
titration
method. Magnesium can be de-
termined similarly by measurements on
specimens diluted 25- to 500-fold
with water. The quantity of urine
required is only 0.1 to 1 ml.
the development by the
Following
author of rapid methods (6, 7)
for the determination of calcium and
magnesium in blood serum by atomic
absorption spectroscopy (5) the method
was applied to the clinically important
determination of these elements in
urine. The techniques developed for
serum analysis had to be modified for
the following reasons:
The calcium and magnesium contents
are much more variable than in blood
serum; the phosphorus content is
variable and sometimes very high; and
in the analysis of urine, which contains
little or no protein, the chemical inter-
ference due to the presence of phos-
phorus is much more pronounced than
in serum analysis, where the high pro-
tein concentration largely compensates
for this interference.
EXPERIMENTAL
Apparatus. The apparatus was
used in the earlier work (#, 6). The
source twin-electrode calcium/
was a
magnesium hollow-cathode tube (made
by Ransley Glass Instruments, Mel-
bourne, Australia) which was run from
a half-wave rectified power supply.
The hollow-cathode power supply and
an inexpensive unit comprising photo-
multiplier power supply, amplifier, rec-
556 · ANALYTICAL CHEMISTRY
for making satisfactory corrections.
LITERATURE CITED
(1) Baar, S.. Clin. Chim. Acta 2, 567-75
(1957).
(2) Chilcote, . E., Wasson, R. D., Clin.
Chem. 4, 200-10 (1958).
(3) Elliott, J. E., Pearson, P. B., J. Lab.
tifier, and meter are made commercially
by Techtron Appliances, South Mel-
bourne, Australia. The light emitted
by the cathode was focused at the
center of the flame into which the
sample to be analyzed was aspirated,
and was then refocused onto the entrance
slit of a Beckman DU monochromator
set to pass the appropriate resonance
line (Ca 4227 A., Mg 2852 A.). The
signal from a 1P28 photomultiplier
behind the exit slit of the monochrom-
ator was amplified by a simple alter-
nating current amplifier, rectified, and
read on a microammeter. By adjusting
the amplifier gain so that a reading of 100
divisions was obtained when distilled
water was aspirated into the flame the
percentage transmission when the
sample was atomized could be read off
directly.
The 10-cm. burner used in the earlier
work (6) tended to distort with the
heat of the air-acetylene flame and was
replaced by one of more massive con-
struction (Figure 1). This burner was
fitted to the spray chamber and atomizer
of a commercial flame photometer
(Evans Electroselenium Ltd., London,
England). The uptake of liquid by
this atomizer was 3.3 ml. per minute.
An air-acetvlene mixture was used, the
consumption of air being about 3.5
that
Clin. Pathol. 28, 208-17 (1957).
(5) Ibid,., pp. 689-93.
(6) Kingsley, G. R., Robnett, 0., Ibid.,
27, 223-30 (1957).
(7) Ibid., 29, 171-5 (1958).
(8) Kingsley, G. R., Schaffert, R. R.,
Anal. Chem. 25, 1738-41 (1953).
Received for review August 15, 1960.
Accepted January 5, 1961.
liters per minute and that of acetylene
about 1.2 liters per minute.
Standard Preparation. All reagents
were of analytical quality, and except
for hydrochloric acid, which was dis-
tilled before use, were not further
purified.
Standard calcium solutions were made
by dilution from a stock solution con-
taining 1000 p.p.m. of calcium made
by dissolving oven-dried calcium car-
bonate in the minimum quantity of
hydrochloric acid and diluting to vol-
ume. Standard magnesium solutions
were made up by dilution of a stock
solution containing 1000 p.p.m. of mag-
nesium, made by dissolving pure mag-
nesium turnings in the minimum quan-
tity of hydrochloric acid and diluting
to volume.
Sample Preparation. Urine was
preserved by the addition of about 3%
of its volume of concentrated hydro-
chloric acid. Some of the specimens,
which had been kept for several weeks,
were centrifuged before measurement
to remove deposits of uric acid, etc.
They were prepared for measurement
by one of the following methods.
(a) Separation of Calcium by Pre-
cipitation as Oxalate. Urine (0.5 to
3 ml., depending on the expected cal-
cium content) was pipetted into a 10-ml.
Figure 1. Isometric
sketch of half the 1 0-cm.
stainless steel burner
dowelled
Two halves are
and screwed together. Meas-
urements are In mm.
centrifuge tube and 1 ml. of an oxalate
buffer (10 ml. of O.lAf oxalic acid +190
ml. of O.lAf ammonium oxalate) was
added. The tube was shaken and al-
lowed to stand overnight, after which
it was centrifuged for 10 minutes, the
supernatant liquid poured off, and the
tube allowed to drain for 5 minutes.
After dissolving the precipitate of cal-
cium oxalate in 1 drop of hydrochloric
acid, sufficient strontium chloride solu-
tion (50,000 p.p.m. of Sr) was added
to give a final concentration of 1000
p.p.m. of strontium when the solution
was made up to a suitable volume (5,
10, or 25 ml.) with water. (The stron-
tium chloride was added to prevent
interference from any phosphate which
might be coprecipitated or adhere to
the calcium oxalate precipitate.) The
solution was measured relative to
standard calcium solutions containing
strontium chloride (1000 p.p.m. of Sr).
(b) Direct Dilution with Lanthanum
Chloride. To 0.2 to 1 ml. of urine was
added enough lanthanum chloride solu-
tion (50,000 p.p.m. of La) to give a
final concentration of 10,000 p.p.m. of
lanthanum when the solution was made
up to a suitable volume (5, 10, or 25
ml.) with water. Any cloudiness de-
veloping on the addition of the lan-
thanum solution was cleared by adding
a drop or two of hydrochloric acid.
The solution was measured relative to Phosphorus interference with calcium absorption in 10-cm. air-
Figure 2.
standard calcium solutions containing
lanthanum chloride (10,000 p.p.m. acetylene flame
of La).
(c) Direct Dilution with Strontium
Chloride. The same as for (b) except
that sufficient strontium chloride was Table I. Comparison of Results of Different Methods for Determination of Calcium
added to give a final concentration of in Urine
10,000 p.p.m. of Sr in the unknown (Mg./lOO ml.)
and standard solutions.
Method
(d) Direct Dilution with Water.
Urine (0.25 to 1 ml.) was diluted with ecimen Phosphorus, Oxalate-
water to a suitable volume (5 to 100 No. Mg./lOO Ml. (a) (b) (c) (e) permanganate
ml.), and the solution measured relative 1 40.8 0.8 0.95
to standard magnesium solutions in 2 34.8 1.1 1.6 1.0 6.85
water alone. 3 36.7 1.4 1.45 1.45 1.3
4 12.8 4.55 4.55 4.7 5.0
(e) Ashing of Samples. Urine (1 ml.) 6.25
was pipetted into a platinum crucible,
5 8.8 6.1 6.1 6.55
6 42.1 13.0 13.1 13.3 13.1
evaporated to dryness on the water 7 28.1 16.5 16.6 16.5 17.3
bath, and ashed at 500° to 550° C. for 8 12.9 16.6 16.8 16.7 16.3
16 hours in a muffie furnace or by 9 5.6 21.3 20.8 21.6 20.6
gently warming with a drop of sulfuric 10 57.7 1.9 1.7 1.6 1.8
acid until ashing was complete; a 11 78.6 2.1 2.1 2.4 2.3
blank measurement was carried out 12 28.7 72.4 73.5 76.0
when using the latter method. The 13 19.2 44.6 43.5 45.9 42.8 44.6
ash was dissolved in a drop or two of 14 95.9 24.9 25.2 24.5 24.7
15 5.0 5.2 5.1 5.6 5.1
hydrochloric acid and made up to a 16 27.5 24.8 25.0 25.0
suitable volume with water containing 17 7.5 7.4 7.1
strontium or lanthanum chloride (10,000 18 24.6 24.5 24.4
p.p.m. of the metal).
Specimens 1-11 are pathological specimens; 12 and 13 are faecal samples, ashed at
550° C. for 24 hours, and extracted with 10% hydrochloric acid; 14-18 are normal urines.
DETERMINATION OF CALCIUM

Investigation of Phosphorus Inter-

ference. In the earlier work (6) it
was shown that the marked chemical
interference with the calcium absorp-
tion caused by the presence of phos-
phorus in the solution being analyzed
could be overcome, for P to Ca
ratios up to about 4, by the addition
of 2500 p.p.m. of strontium or 10,000
p.p.m. of the disodium salt of (ethyl-
enedinitrilo)tetracetic acid. Since
some of the urines to be analyzed had
P to Ca ratios approaching 40, further
study of the phosphorus interference
was clearly necessary. Figure 2 shows
how the depression of the calcium ab-
sorption by increasing quantities of
phosphorus (as H3PO4) is controlled by
the addition of 1000 and 10,000 p.p.m.
of strontium (as SrCL), 10,000 p.p.m.
of lanthanum (as LaCL), and 10,000
p.p.m. of the disodium salt of (ethylene-
dinitrilo) tetracetic acid. Strontium
and lanthanum at concentrations of
10,000 p.p.m. are the most effective
suppressors of the interference at high
phosphorus concentrations, and lan-
thanum has an important advantage
over strontium in that it does not itself
lower the calcium absorption.
It should be possible, then, to esti-

vón 33, NO. 4, APRIL 1961 · 557

 measurement of a dilute solution after
Table II. Effect of Dilution on Calcium Determination the addition of 10,000 p.p.m. of
(Ca, mg./lOO ml.) strontium or lanthanum: the results
Method
obtained agree with those found by
Dilution precipitation of the calcium as oxalate
of Urine (a) (b) (c) and estimation of this precipitate by the
No. 16 No. 17 No. 18 No. 16 oxalate-permanganate method. The in-
ternal consistency of the atomic ab-
1:10 25.5 7.5 25.0
1:20 24.8 7.5 24.6 25.4 sorption method is also good, since the
1:40 24.8 24.3 24.4 results obtained by direct dilution
1:100 24.9 agree with those obtained by atomic
absorption measurements on the cal-
cium after separation from the urine
Table III. Results of Recovery Experiments on Determination of Calcium in Urine by precipitation with oxalate. The
Ca, P.P.M. results of dilution and recovery experi-
Concentra- ments (Tables II and III) are also
Specimen tion in Recovery satisfactory.
No. solution Added Total Recovered % The repeatability and reproducibility
Method (a) of the method are illustrated by the
1 3.15 4.7 7.85 7.5 95.5 results showm in Table IV. Six repli-
6 4.05 3.1 7.15 7.25 101.5 cate 1-ml. samples of urine w'ere diluted
6 4.1 3.1 7.2 7.2 100.0 to 10 ml. with lanthanum chloride
14 5.6 8.0 13.6 13.6 100.0
16 6.2 5.0 11.2 11.05 98.5 solution and measured in the manner
recommended below'. The same solu-
Method (b)
tions w'ere remeasured three hours later,
1 1.40 2.35 3.75 3.7 98.5
6 10.1 7.7 17.8 18.05 101.5 using a different calcium hollow-cathode
18 6.3 5.0 11.3 11.4 101 tube and a different atomizer. The
18 6.3 10.0 16.3 16.6 102 coefficient of variation for a single de-
termination was 1.1% and the re-
producibility on remeasurement was
about the same.
mate calcium in urine by measurement
Table IV. Repeatability and Repro· of solutions diluted with either stron-
ducibility of Method (b) for Determl· tium or lanthanum chlorides at the
DETERMINATION OF MAGNESIUM
nation of Calcium in Urine above concentration and calibration of
(mg./lOO ml.) the instrument with standard calcium Allan {1) found that in the air-acety-
solutions containing this same con- lene flame the absorption of magnesium
Ca Content
Replicate Ca Content (measured 3 centration of strontium or lanthanum. is not affected by the presence of
No. Specimen 17a hours later) Experiments showed that in such solu- sodium or phosphorus at the concentra-
1 7.04 7.13 tions sodium and potassium, even in tions at which these occur in biological
2 7.04 7.19 concentrations 200 times that of the materials, and this work show's that
3 7.11 7.31 calcium, had no effect on the absorption
4 7.11 7.13 the magnesium content of urine can be
due to this metal. determined by measurement of samples
5 7.11 7.26
6 7.25 7.26 Results. Table I shows that the diluted directly with water. The ab-
Av. 7.11 7.21 calcium content of urine, even where sence of interference from the organic
Std. dev. 0.08 0.08 the P to Ca ratio is high, can be constituents of the urine is shown by
satisfactorily determined by direct the results in Table V, which records
the magnesium contents of four widely
different specimens measured on directly
diluted samples and also on solutions
Table V. Comparison of Different Methods for Determination of Magnesium of the ashed materials. The internal
in Urine
consistency of the atomic absorption
(mg./lOO ml.) method is demonstrated by the dilution
Atomic Absorption and recovery experiments shown in
Specimen Direct Titan Yellow Tables VI and VII.
No. dilution Ashed Direct Ashed The difficulty of selecting a suitable
7 5.41 5.40 chemical method of analysis for checking
10 0.22 0.23 the results of the atomic absorption
13 26.0 25.3 27.1 28.6 method was discussed in the earlier
14 20.0 19.5 17.2 18.7
w'ork (7), but as a matter of interest
several specimens of urine were an-
Table VI. Effect of Dilution on Magnesium Determination
alyzed for magnesium by the modifica-
tion of the Titan Yellow' method re-
(mg./lOO ml.) cently described by Fletcher et al. (S).
Specimen No. 9 Specimen No. 14 Specimen No. 16 Some of the results are shown in Table
Mg Mg Mg V.
Dilution content Dilution Content Dilution content Wacker and Vallee (4) consider the
1:25 5.00 1:100 19.5 1:50 9.68 Titan Yellow method unreliable, and
1:33 4.81 1:200 20.3 1:100 9.51 the present work tends to support their
1:50 5.00 1:400 20.4 1:200 9.62
1:100 4.97 view. Slightly different results were
1:200 5.12 obtained depending on whether, the
sample was measured directly or after

558 · ANALYTICAL CHEMISTRY

ashing; furthermore, recovery of mag-
nesium added to the sample often Table VII. Results of Recovery Experiments on Determination of Magnesium
differed markedly from 100%. in Urine
Mg, P.P.M.
Concentra-
RECOMMENDED METHOD Specimen tion in Recovery
No. solution Added Total Recovered %
Determination of Calcium. Using 1 0.55 0.29 0.84 0.87 103.5
a stock solution of lanthanum chloride 6 0.74 0.31 1.05 1.05 100
9 0.74 0.29 1.03 1.04 101
containing 50,000 p.p.m. of La, pre- 9 0.71 0.29 1.00 0.99 99
pare standard solutions of 0, 5, 10, 15, 14 1.02 0.50 1.52 1.52 100
and 20 p.p.m. of Ca containing also 14 1.02 1.00 2.02 2.04 101
10.000 p.p.m. of La. 16 0.96 0.80 1.76 1.77 100.5
Prepare a solution of each urine . 100.7
specimen such that its calcium con-
centration lies in the 5- to 20-p.p.m.
range, adding enough lanthanum stock
solution to give a final concentration of and read off the concentration of the and calcium contents by chemical
10.000 p.p.m. of La, and clearing any sample solutions. The absorbance-con- methods.
cloudiness by adding a drop or two of centration curve is almost a straight
hydrochloric acid. (Since the calcium line. LITERATURE CITED
content of urine is so variable it may Determination of Magnesium. Pre-
be advisable to determine the degree (1) Allan, J. E., Analyst 83, 466 (1958).
pare standards containing 0.5, 1.0, 1.5, (2) Box, G. F., Walsh, A., Spectrochim.
of dilution required by roughly measur- and 2.0 p.p.m. of Mg and dilute the Acta 16, 255 (1960).
ing the absorption of 0.5 ml. of urine urine specimens so that the magnesium (3) Fletcher, R. F., Henly, A. A., Sam-
and 2 ml. of stock La solution diluted concentration lies between 0.5 and 2 mons, H. G., Squire, J. R., Lancet p.
to 10 ml.) p.p.m. Measure standards and sam- 522 (March 5th, 1960).
Measure the absorptions of the solu- ples in the same way as for calcium. (4) Wacker, W. E., Vallee, B. L., New
tions in the following order: standards, England J. Med. 259, 431 (1958).
samples, standards, samples, standards. (5) Walsh, A., Spectrochim. Acta 7, 108
Each reading takes about 7 seconds and ACKNOWLEDGMENT (1955).
(6) Willis, J. B., Ibid., 16, 259 (1960).
requires approximately 0.3 ml. of solu- The writer thanks Roger Melick, (7) Ibid., p. 273 (1960).
tion.
Average the absorption values for who supplied the samples of pathological Received for review October 19, 1960.
each solution, plot the calibration curve urine and measured their phosphorus Accepted December 27, 1960.

Extension of the Porter-Silber Reaction to

17-Deoxy-alpha-ketolic Steroids
MARVIN L. LEWBART and VERNON R. MATTOX

Mayo Clinic and Mayo Foundation, Rochester, Minn.

A method for the quantitative
microdetermination of 17-deoxy-a-
ketolic steroids is described. The
method consists of a preliminary
cupric acetate oxidation of such com-
pounds to the corresponding glyoxals
followed by treatment with the Porter-
Silber reagent. For all a-ketolic
steroids tested a strict adherence to
Beer's law was obtained.
Porter-Silber reaction to include such
compounds. This paper presents a
method for the microestimation of pure
ketols employing cupric acetate oxi-
dation followed by treatment with
the Porter-Silber reagent. In addition,
a procedure for converting 17-OH-
ketols and ketols to glyoxals on paper
chromatograms is described.
MATERIALS
prepared by mixing two parts of 7:3
H2S04-H20 containing 0.5 mg. per ml.
of phenylhydrazine hydrochloride and
one part of 95% ethyl alcohol.
0.005.¥ Cupric Acetate Solution. One
milligram of Cu(0Ac)2.H20 (Mallinc-
krodt) per milliliter of methanol.
Solutions retain their full oxidizing
activity for at least 2 weeks despite
some color change and precipitation.
Abbreviations Used. The following
abbreviations are employed: THA =

Methanol. Distilled from anhydrous

1950 Porter and Silber (7) de- 3 ,21 dihydroxypregnane
-

11,20- -

potassium carbonate.
IN
scribed a color reaction for 17,21-
Phenylhydrazine Hydrochloride. Re- dione; THB 3a,ll3,21-trihydroxy-
=

dihydroxy-20-ketosteroids (17-OH-ke- crystallized three to four times from pregnan-20-one; THQ 3 ,21-=

tols) which has since proved to be of 95% ethyl alcohol. dihydroxypregnan-20-one; THE 3a,- =

great value in the quantitative deter- Porter-Silber Reagent A. For quan- 17,21 trihvdroxypregnane-11,20-dione;
-

mination of these substances. Al- titative analysis. This was prepared THF 3 , 11 ß, 17,21 -t etrahy d roxy preg-
=

though 21-hydroxy-20-ketosteroids (ke- by dissolving 50 mg. of phenylhydrazine nan-20-one; THS 3a,17,21-trihy-

=

tols) give no color with the reagent, hydrochloride in 100 ml. of an 8:2 droxypregnan-20-one; 16- 3 ,- =

their corresponding 21-aldehydes or mixture of concentrated H2S04 and

H20. For color development, 2 ml.
21-dihy droxy-16- pregnene-11,20-dione;
glyoxals react even more rapidly than of this solution was freshly prepared compound 35,21-dihydroxyallo-
=

17-OH-ketols (8). Since ketols can be
oxidized readily in high yield to glyoxals
by reaction with cupric acetate (3,9),
and mixed with 1 ml. of methanol pregnane-11,20-dione; substance R
containing the dissolved steroid.
=
3ß,11ß,2 l-trihydroxyallopregnan-20-
Porter-Silber Reagent B. For use one; THA glyoxal hydrate 3 a- =

it has been possible to extend the on paper chromatograms. This was hydroxy-1 l,20-dioxopregnan-21-al hy-

VOL. 33, NO. 4, APRIL 1961 · 559