The Coleman flame photometer was not satisfactory for urinary calcium determinations. Effect of Hemolysis and Bilirubin. Slight hemolysis and bilirubin concentrations as high as 4 mg. % had no effect on the rate or completion of color development of the NFRcalcium color complex. At higher
concentrations of bilirubin and greater hemolysis blanks could not be used for making satisfactory corrections. LITERATURE CITED
(1) Baar, s.. Clin. Chim. Acta 2, 567-75 (1957). (2) Chilcote, M. E., Wasson, R. D., C h . Chem. 4, 200-10 (1958). (3) Elliott, J. E., Pearson, P. B., J . Lab.
Clin. Med. 31, 1262-6 (1946).
ti,~o~17$l~a,,””*
( 4 ~ ~ ~ ~ ~ a t ~ ; l , ( 5 ) Ihid., pp. 689-93. (6) Kingsley, G. R., Robnett, O., Ibid.,
27, 223-30 (1957). (7) Ihid., 29, 171-5 (1958). (8) Kingsley, G. R., Schaffert, R. R., API’AL.CHEM.25, 1738-41 (1953).
RECEIVED for review August 15, 1960. Accepted January 5, 1961.
Determination of Calcium and Magnesium in Urine by Atomic Absorption Spectroscopy J.
B. WlLLlS
Division of Chemical Physics, C. S. I . R. 0. Chemical Research laboratories, Melbourne, Australia
b The calcium content of urine may b e determined b y atomic absorption measurement of specimens diluted 5to 50-fold with a solution of either lanthanum chloride or strontium chloride containing 1 % b y weight of the metal. The solution is sprayed into an airacetylene flame. The values obtained agree well with those obtained b y the permanganate titration oxalate method. Magnesium can b e determined similarly b y measurements on specimens diluted 25- to 500-fold with water. The quantity of urine required is only 0.1 to 1 ml.
-
F
the development by the author of rapid methods (6, 7) for the determination of calcium and magnesium in blood serum by atomic absorption spectroscopy (6) 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 interference due to the presence of phosphorus is much more pronounced than in serum analysis, where the high protein concentration largely compensates for this interference. OLLOWING
EXPERIMENTAL
Apparatus. The apparatus was that used in the earlier work (8, 6). The source was a twin-electrode calcium/ magnesium hollow-cathode tube (made by Ransley Glass Instruments, Melbourne, Australia) which was run from a half-wave rectified power supply. The hollow-cathode power supply and an inexpensive unit comprising photomultiplier power supply, amplifier, rec-
556
ANALYTICAL CHEMISTRY
tifier, and meter are made commercially by Techtron Appliances, South Melbourne, Australia. The light emitted by the cathode was focused a t 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 monochromator was amplified by a simple alternating 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 samde 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 construction (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-acetylene mixture was used, the consumption of air being about 3.5
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 distilled before use, were not further purified. Standard calcium solutions were made by dilution from a stock solution containing 1000 p.p.m. of calcium made by dissolving oren-dried calcium carbonate in the minimum quantity of hydrochloric acid and diluting to volume. Standard niagnesium solutions were made up by dilution of a stock solution containing 1000 p.p.m. of magnesium, made by dissolving pure magnesium turnings in the minimum quantity of hydrochloric acid and diluting to volume. Sample Preparation. Urine was preserved by the addition of about 3% of its volume of concentrated hydrochloric 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 Precipitation as Oxalate. Urine (0.5 to 3 ml., depending on the expected calcium content) was pipetted into a 10-ml.
Figure 1. Isometric sketch of half the 1 0-cm. stainless steel burner Two halves are dowelled and screwed together. Mearurements are in rnrn.
centrifuge tube and 1 ml. of an oxalate buffer (10 ml. of O.1M oxalic acid +190 ml. of 0.lM ammonium oxalate) was added. The tube was shaken and allowed 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 calcium oxalate in l drop of hydrochloric acid, sufficient strontium chloride solution (50,000 p.p.m. of Sr) was added to give a final concentration of 1000 p.p.m. of strontium mhen the solution mas made up to a suitable volume (5, 10, or 25 ml.) with water. (The strontium 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 solution (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 t o a suitable volume (5, 10, or 25 ml.) with mater. Any cloudiness developing on the addition of the lanthanum solution was cleared by adding a drop or two of hydrochloric acid. The solution was measured relative to standard calcium solutions containing lanthanum chloride (10,000 p.p.m. of La). (e) Direct Dilution with Strontium Chloride. The same as for (b) except that sufficient strontium chloride was added to give a final concentration of 10,000 p.p.m. of Sr in the unknown and standard solutions. (d) Direct Dilution with Water. Urine (0.25 to 1 ml.) was diluted with water to a suitable volume ( 5 to 100 ml.), and the solution measured relative to standard magnesium solutions in mater alone. (e) Ashing of Samples. Urine (1 ml.) was pipetted into a platinum crucible, evaporated to dryness on the water bath, and ashed a t 500' to 550' C. for 16 hours in a muffle furnace or by gently warming with a drop of sulfuric acid until ashing was complete; a blank measurement was carried out when using the latter method. The ash was dissolved in a drop or two of hydrochloric acid and made up to a suitable volume with water containing strontium or lanthanum chloride (10,000 p.p.m. of the metal), DETERMINATION
OF
\ca
1Opprn.
rO
IoOOOppm
ca l o p p m + E D T A ca
IO
0
100 PHOSPHORUS,
Figure 2.
Table I.
iOOOOpPm
i o p p m + sr loo00 p p m
1000
ppm
Phosphorus interference with calcium absorption in 1 0-cm. airacetylene flame
Comparison of Results of Different Methods for Determination of Calcium in Urine
(Mg./100 ml.)
Method Specimen Phosphorus, Mg./100 M1. KO. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
( a)
40.8 34.8 36.7 12.8 8.8 42.1 28.1 12.9 5.6 57.7 78.6 28.7 19.2 95.9
13.0 16.5 16.6 21.3
27.5
5.0 24.8
... ...
0.8
1.1 1.4 4.55
...
...
( b)
(C )
(e)
...
0.95 1.0 1.45 4.7 6.1 13.3 16.5 16.7 21.6 1.6 2.4 76.0 45.9
...
1.0 1.45 4.55 6.1 13.1 16.6 16.8 20.8
5.2
...
7.5 24.6
5.1 25.0
...
...
... ...
6.25
...
... ... ... ... ... ...
42.8 5.0
...
7.4 24.5
Oxalatepermanganate
...
0.85 1.3 5.0 6.55 13.1 17.3 16.3 20.6 1.8 2.3
...
44.0 24.7 5.1 25.0 7.1 24.4
... ... 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
CALCIUM
Investigation of Phosphorus Interference. I n the earlier work (6) it was shown t h a t the marked chemical interference with the calcium absorption caused by the presence of phosphorus 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 (ethylenedinitri1o)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 absorption by increasing quantities of phosphorus (as HsP04) is controlled by the addition of 1000 and 10,000 p.p.m. of strontium (as SrClZ), 10,000 p.p.m. of lanthanum (as LaC13), and 10,000
p.p.m. of the disodium salt of (ethylenedinitri1o)tetracetic acid. Strontium and lanthanum a t concentrations of 10,000 p.p.m. are the most effective suppressors of the interference a t high phosphorus concentrations, and lanthanum has an important advantage over strontium in that it does not itself lower the calcium absorption. It should be possible, then, to estiVOL 33, NO. 4, APRIL 1961
557
Table II.
Effect of Dilution on Calcium Determination (Ca, mg./100 ml.)
Method Dilution of Urine
(a) No. 16
1:lO 1:20 1:40 1:lOO Table 111.
(b)
No. 17
25.5 24.8 24 8
7.5 7.5
...
24.6 24.3 24.9
...
...
( C)
No. 18
...
No. 16 25.0 25.4 24.4
...
Results of Recovery Experiments on Determination of Calcium in Urine
Ca, P.P.M. No.
Concentration in solution
1 6 6 14 16
3.15 4.05 4.1 5 6 6 2
1 6 18 18
1.40 10.1 6.3 6.3
Specimen
Recovery Added Total Method (a) 4.7 3.1 3 1 8 0 5.0
7.85 7.15 7.2 13 6 11.2 Method (b) 2.35 3.75 7.7 17.8 11.3 5.0 10.0 16.3
Table IV. Repeatability and Reproducibility of Method (b) for Determination of Calcium in Urine (mg./100 ml.)
,
Ca Content Replicate Ca Content (measured 3 No. Specimen 17a hours later) 1 2 3
7.04 7.04 7.11 4 7.11 0 7.11 6 7.25 AV. 7.11 Std. dev. 0.08
Table V.
Specimen NO. 7 10 13 14
7.13 7,19 i.31
7.13 7.26 7.26 7.21 0 08
Recovered
70
7.5 7.25 7.2 13 6 11 05
95.5 101.5 100.0 100 0 98 5
3.7 18.05 11.4 16.6
98.5 101.5 101 102
mate calcium in urine by measurement of solutions diluted with either strontium or lanthanum chlorides a t the above concentration and calibration of the instrument with standard calcium solutions containing this same concentration of strontium or lanthanum. Experiments showed that in such solutions sodium and potassium, even in concentrations 200 times thxt of the calcium, had no effect on the absor;)tioii due to this metal. Results. Table I shows t h a t the calcium content of urine, even nhere the P t o Ca ratio is high, can be satisfactorily determined by diiei. t
Comparison o f Different Methods for Determination of Magnesium in Urine (mg./100 ml.)
Atomic hbsorption Direct dilution Ashed 5.40 0.23 25.3 19.5
6 41
0.22 26.0 20.0 Table VI.
Specimen No. 9
Titan Yellow Direct Ashed... 27.1 17.2
Effect of Dilution on Magnesium Determination (mg./100 ml.) Specimen KO. 14 Specimen X o . 16
Dilution
content
Dilution
Mg Content
Dilution
1:25 1:33 1:50 1: 100 1:200
5 00 4 81 5 00 4 97 5 12
1:lOO 1:200 1:400
19 5 20 3 20 4
1:50 1: 100 1 :200
558
MI3
... 28.6 18.7
ANALYTICAL CHEMISTRY
Mg content 9 68
9 51
9 62
measurement of a dilute solution after the addition of 10,000 p.p.m. of strontium or lanthanum: the results obtained agree n i t h those found by precipitation of the calcium as oxalate and estimation of this precipitate by the oxalate-permanganate method. The internal consistency of the atomic absorption method is also good, since the results obtained by direct dilution agree with those obtained by atomic absorption measurements on the calcium after separation from the urine by precipitation with oxalate. The results of dilution and recovery experiments (Tables I1 and 111) are also satisfactory. The repeatability and reproducibility of the method are illustrated by the results shown in Table IV. Siu replicate 1-ml, samples of urine were diluted to 10 ml. with lanthanum chloride solution and measured in the manner recommended below. The same solutions were remeasured three hours later, using a different calcium hollow-cathode tube and a different atomizer. The coefficient of variation for a single determination F m s 1.17' and the reproducibility on remeasurement n as about the same.
DETERMINATION
OF
MAGNESIUM
Allan ( 1 ) found that in the air-acetylene flame the absorption of magnesium is not affected by the presence of sodium or phosphorus a t the concentrations a t which these occur in biological materials, and this n ork shows that the magnesium content of urine can be determined by measurement of samples diluted directly with mater. The absence of interference from the organic constituents of the urine is shown by the results in Table V, which records the magnesium contents of four widely different specimens measured on directly diluted samples and also on solutions of the ashed materials. The internal consistency of the atomic absorption method is demonstrated by the dilution and recovery experiments shown in Tables VI and VII. The difficulty of selecting a suitable chemical method of analysis for checking the results of the atomic absorption method was discussed in the earlier work (Y), but as a matter of interest several specimens of urine m r e analyzed for magnesium by the modification of the Titan Yellow method recently described by Fletcher et al. ( 3 ) . Some of the results are shown in Table
V.
Wacker and Vallee (4) consider the Titan Yellow- method unreliable, and the present work tends to support their view. Slightly different results were obtained depending on whether the sample was measured directly or after
asking; furthermore, recovery of magnesium added to the sample often differed markedly from 100%. RECOMMENDED METHOD
Table VII.
Specimen S O .
Determination of Calcium. Using a stock solution of lanthanum chloride
containing 50,000 p.p.m. of La, prepare standard solutions of 0, 5, 10, 15, and 20 p.p.m. of Ca containing also 10.000 p.p.m. of La. Prepare a solution of each urine specimen such that its calcium concentration lies in the 5- t o 20-p.p.m. range, adding enough lanthanum stock solution t o give a final concentration of 10,000 p.p.m. of La, and clearing any cloudiness by adding a drop or two of hydrochloric acid. (Since the calcium content of urine is so variable it may be advisable to determine the degree of dilution required by roughly measuring the absorption of 0.5 ml. of urine and 2 ml. of stock La solution diluted to 10 ml.) Measure the absorptions of the solutions in the following order: standards, samples, standards, samples, standards. Each reading takes about 7 seconds and rcxquires approximately 0.3 ml. of solution. Average the absorption values for each solution, plot the calibration curve
1
6 9 9
14
14 16
Results of Recovery Experiments on Determination of Magnesium in Urine
Concentration in solution 0..55 0.74 0.74 0.71
1.02 1.02 0.96
Added 0.29 0.31 0.29 0.29 0.50 1.00 0.80
and read off the concentration of the sample solutions. The absorbance-concentration curve is almost a straight line. Determination of Magnesium. Prepare standards containing 0.5, 1.0, 1.5. and 2.0 p.p.m. of Mg and dilute the urine specimens so that the magnesium concentration lies between 0.5 and 2 p.p.m. Measure standards and samples in the same way as for calcium.
Total
Recovered
0.84
0.87
i.05
1.03 1.00 1.52 2.02 1.76
1.05 1.04
0.99 1.52 2.04 1.77
Recovery % 103.5 100 101 99 100 101 100.5 Av. 100.7
and calcium contents by methods.
chemical
LITERATURE CITED
(1) Allan, J. E., Analyst 83, 466 (1958). (2) Box, G. F., Walsh, A., Spectrochim. Acta 16, 255 (1960). (3) Fletcher, R. F., Henly, A. A., Sammons. H. G.. Sauire. J. R.. Lancet D.
. .
ACKNOWLEDGMEN'I
The writer thanks Roger Melick, who supplied the samples of pathological urine and measured their phosphorus
(1955). (6) Willis, J. B., Zbid., 16, 259 (1960). ( 7 ) Ibid., p. 273 (1960). RECEIVED for review October 19, 1960. Accepted December 27, 1960.
Extens io n O f the Porter-Si1ber Reaction to 17-Deoxy-aIpha- keto1ic Steroids MARVIN
L. LEWBART
and VERNON R. MATTOX
Mayo Clinic and Mayo Foundation, Rochester, Minn.
b A method for the quantitative microdetermination of 17-deoxy-aketolic steroids i s described. The method consists of a preliminary cupric acetate oxidation of such compounds to the corresponding glyoxals followed b y treatment with the PorterSilber 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 oxidation followed by treatment with the Porter-Silber reagent. I n addition, a procedure for converting 17-OHketols and ketols to glyoxals on paper chromatograms is described. MATERIALS
I
1950 Porter and Silber (7) described a color reaction for 17,21dihydroxy-20-ketosteroids (17-OH-ketols) rvhich has since proved to be of great valur in the quantitative determination of these substances. *kltliough 21-hydroxy-20-ketosteroids (ketols) give no color a i t h the reagent, their corresponding 21-aldehydes or glyoxals react even more rapidly than 17-OH-kptols (8). Since ketols can be oxidized readily in high yield to glyoxals by reaction with cupric acetate (3,9), it has been possible to extend the N
llethanol. Distilled from anhydrous potassium carbonate. Phenylhydrazine Hydrochloride. Recrystallized three to four times from 95% ethyl alcohol. Porter-Silber Reagent A. For quantitative analysis. This was prepared by dissolving 50 mg. of phenylhydrazine hydrochloride in 100 ml. of an 8:2 mixture of concentrated HQS04 and HzO. For color development, 2 nil. of this solution freshly prepared and mixed with 1 ml. of methanol containing the dissolved steroid. Porter-Silber Reagent B. For use on paper chromatograms. This was
prepared by mixing t n o parts of 7 : 3 H2SOa-H20 containing 0.5 mg. per ml. of phenylhydrazine hydrochloride and one part of 95% ethyl alcohol. 0.005M Cupric Acetate Solution. One milligram of CU(OAC)~ H 2 0 (llallinckrodt) per milliliter of methanol. Solutions retain their full oxidizing activity for a t least 2 15-eeks despite some color change and precipitation. Abbreviations Used. The following abbreviations are employed: 'I'HA4 = 3 ~ ~ 2- 1dihydroxypregnane - 11,20dione; T H B = 3 a , l l B,21-trihydroxypregnan-20-one; THQ = 3cq21dihydrosypregnan-20-one; T H E = 3a,17,21- trihydroxypregnane - 11,20-dione; T H F = 3n,llp,l7,21-tetrahydroxypregnan-20-one; THS = 3~~,17,21-trihydroxypregnan-20-one; AlG-THA = 3a,21-dihydroxy-l B-pregnene - 11,2O-dione; compound H = 3p,21-dihydroxyallopregnane-11,20-dione; substance R = 3p711p,21-trihydroxyallopregnan-20one; THA glyoxal hydrate = 3ahydroxy-11,2O-dioxopregnan-21-a1 hyVOL. 33, NO. 4, APRIL 1961
559
