are shown in Table VII. Each sample contained 250 mg. of gelatin dissolved in 25 ml. of water. These were titrated to p H 11.5 with 0.3000N Ba(OH)2, then excess Ba(OH)2 amounting to 18.000 X equivalent was added to each and the preparations back- titrated to p H 11.5 with 0.2000AT CUSOI. This protein utilizes 1.887 * 0.025 (S.D) milliequivalents of alkali per gram in excess of that required to neutralize ordinary acidic groups and to account for enolic groups stabilized by reaction with copper. The proteins vary somewhat as to the amount of alkali they can consume in excess of these requirements, but, once this is known, the amount of protein in a sample can be measured by titrating it t o p H 11.5 to neutralize the ordinary acidic groups, adding a known and ex- cessive amount of barium hydroxide, and back-titrating to p H 11.5 with standard copper sulfate. The weight is calcu- lated by finding the difference, in equir-

alents, between the amount of hy- droxide used above p H 11.5 and the amount of copper and multiplying this times the appropriate factor. This method should prove useful with protein solutions that are too highly colored or too turbid to be suitable for spectro- photometric measurement.

LITERATURE CITED

(1) Block, R. J., Science 108, 608-9 (1948).

(2) Block, R. J., Bolling, Diana, “The Amino Acid Composition of Proteins and Foods,” 2nd ed., pp. 3-34, C. C Thomas, Springfield, Ill., 1951.

(3) Block, R. J., We,i;s, Kathryn, “Amino Acid Handbook, C. C Thomas, Springfield, Ill., 1956.

(4) Datta, S. P., Rabin, B. R., Biochim. et

(5) Dobbie, Hazel, Kermack, W. O., Biochem. J . 59, 257-64 (1955).

(6) Freeman, H. C., Smith, J. E. W. L., Taylor, J. C., Nature 184, 707-10

(7) Gurd, F. R. X., Wilcos, P. E., d d -

Btophys. Acta 19, 572-4 (1956).

(1959).

vances in Protein Chem. 11, 311-427 (1956).

(8) How, P. E., J. Bid. Chem. 49, 93-107 (1921).

(9) Kober, P. A,, Ham, A. B., J . Am. Chem. SOC. 38, 457-72 (1916).

(10) McDonald, H. J., “Ionography,” p. 36, Yearbook Publishers, Chicago, Ill., 1955.

(11) Mehl, J. W., Pacovska, E., Winzler,

(12) Plekhan, M. I., Russianora, N. D., Zhur. ObshcheZ Khim. 23, 512-18

R. J., J . Bioi. C h a . 177, 13-21 (1949).

(1953). (13) Spector, W. S., “Handbook of

Biological Data,” p. 24, W. B. Saunders, PhiladelDhia. Pa.. 1956.

(14) Strichand, R. D., Mack, P. A., Childs, w. A,, ANAL. CHEhi. 32, 430-6 (1960).

(15) Tshugaev, L., Ber. deut. chem. Ges.

RECEIVED for reviex September 6, 1960. Accepted November 14, 1960. Division of Biological Chemistry, 138th Meeting, ACS, Sew York, K. Y., September 1960. Investigation supported in part by a research grant, H2100, from the National Heart Institute, Sational Institutes of Health, Public Health Service, Depart- ment of Health, Education, and Welfare‘

40, 1973-80 (1907).

Investigation of Nuclear Fast Red Method of Baar for Direct Spectrophotometric Determination of Calcium in Serum, Urine, and Spinal Fluid G. R. KINGSLEY and OZlE ROBNETT Clinical Biochemistry laboratory, Veterans Administration Center, and Department of Physiological Chemistry, School of Medicine, University of California, los Angeles 24, Calif.

The optimum conditions for the use of purified nuclear fast red (NFR) were investigated for the determino- tion of calcium in small amounts of serum, urine, and spinal fluid by a simple, direct, and rapid (5 to 10 minutes) spectrophotometric method with well known spectrophotometers. The analytical results obtained with this method for the determination of calcium in biological specimens were in good agreement with those of established methods.

DIRECT method for determining A calcium in serum and urine which does not require the removal of proteins, the precipitation of calcium as oxalate, or a tedious titration procedure, is very desirable as a clinical procedure, especially where speed is desired and large numbers of determinations are made. Calcium methods of this kind have been reported by Kingsley and Robnett (6, 7 ) , Baar (I), and Chilcote and Wasson (2) . The method of Kingsley and Robnett required a dye, disodium - 1 - hydroxy - 4 - chloro - 2,2-

diazobenzene - 1,8 - hydroxynaphtha- lene-3,6-disulfonic acid, which binds calcium to form a complex with less light absorption in solution than the original dye. This condition requires setting the unreacted dye as a blank a t the greatest absorbance reading per- missible for the photometer and then reading the decreasing densities of specimens to measure the amount of calcium present. This situation places a maximum “load” on the electronic system of the photometer which may give nonlinear results if the photometer is not operating efficiently. Although these requirements for proper use of the method have discouraged more general acceptance, this method has been very satisfactory in our hands.

Chilcote and Wasson (2) employed ammonium purpurate for spectrophoto- metric calcium determination, which provided a color that was read in the conventional manner on the spectro- photometer. This method, however, has three principal defects: instability of t he ammonium purpurate standard, greater temperature sensitivity of the color produced, and the requirement

that the color be developed a t 4’ to 15’ C.

The chloranilic acid calcium method of Ferro and Ham (4, 5 ) is not simple, rapid, or direct, as i t requires a large sample of serum, precipitation of pro- teins, washing with isopropyl alcohol, centrifugation, resuspension of pre- cipitate, resolution n-ith EDTA, etc. This procedure requires a t least 60 minutes to perform.

The method of Baar ( 1 ) in which nuclear fast red (NFR) was used, ap- parently avoids the objectional features of the other direct calcium methods cited above. However, one difficulty is encountered: KFR obtained as a solid from commercial sources requires further purification to remove impurities as described by Baar ( 1 ) before i t is satisfactory for the determination of calcium.

The effects of variations of time, tem- perature, concentration of reagents, interference of nonspecific substances, etc., are evaluated to determine the optimum conditions for the use of NFR. We believe these modifications have improved Baar’s original KFR calcium method considerably.

552 0 ANALYTICAL CHEMISTRY

MATERIALS AND METHODS

Reagents. SUCLEAR FAST RED (NFR) ( 1 ) is purified as follows: Dis- solve 5 grams of commercial IYFR in 250 ml. of warm 50y0 ethyl alcohol and cool a t 15' C. for 36 hours. Filter on a Buchner funnel, wash the precipitate with ethyl alcohol until washings are colorless, and finally wash with 100 ml. of ether. Dry over phosphorus pent- oxide. Unrefined SFR is obtainable from George T. Gurr, Ltd., London, S.K. 6, England, or Borden Chemical Co., 5000 Langdon St., Philadelphia 24, Pa. A purified stabilized stock nuclear fast red solution, Fsst R, ready for use may be obtained from I<.I\!t.W. Science Search, Inc., 10866 La Grange Ave., Los Angeles 25, Calif.

STOCK NFR SOLUTIOX. Dissolve 100 nig. of purified N F R in 100 ml. of car- bonste-free triple-distilled water. Let stand for 48 hours and filter off any in- soluble material. This reagent should be stable for several weeks if re- frigerated.

KORKISG SFR SOLUTION. Dilute 10 nil. of stock N F R solution to 50 ml. with carbonate-free 0.1N NaOH (pre- psre fresh before using). If reagent blank fadw, add 116 mg. of (low calcium) bovine albumin fraction V to this re- agent.

STOCK STANDARD CALCIUM CAR- BOKATE SOLUTIOK. Keigh 0.0500 gram of Iceland spar and dissolve in a mini- mum quantity of concentrated HCl. Boil gently to drive off COz. Dilute to 100 ml. with COrfree distilled water. 1 nil. = 200 pg. of calcium. (Spectro- graphically pure CaC03 may also be used.)

Add 1, 2, 3, 4, 5, 6, and 7 ml. of stock standard calcium carbonate solution to 10-ml. volu- metric flasks and dilute to volume with Corfree distilled water. Analyze 0.2 ml. of each of these dilutions as directed in the procedure for t he analysis of serum. Carry out color development during standardization a t a constant 20' t o 25" C. These dilute standards are equivalent t o 2, 4, 6, 8, 10, 12, and 14 mg. of calcium per 100 ml. of serum. Prepare a standardization curve by plotting the per cent transmittance against the concentration of calcium on semilogarithmic paper.

Procedure. SERubi . Add 0.2 ml. of serum with a pipet (calibrated to contain 0.20 i 0.001 ml.) t o a cuvette containing 6 ml. of working N F R solution maintained a t the tempera- ture of standardization (20" t o 25" C.).

K i p e t h e excess serum from the outside of the t ip of the pipet prior to inserting i t into the solution of dye. Wash the pipet (three times) with the dye solution, returning each wash to the cuvette. Prepare a blank by adding 0.2 ml. of distilled water to 6 ml. of the working solution of N F R as described or serum. Permit the cuvette to stand

for 5 minutes for development of color. The color is stable for 30 minutes. Read the absorbance of the specimens and standards against the blank set at 100% T at the optimum absorbance

Standardization.

of the spectrophotometer (575 for Cole- man, Jr., spectrophotometer),

If the serum is lipemic or contains a visible amount of lipides, shake the final NFR-calcium reaction mixture vigorously for 5 seconds with 3 ml. of ethyl ether and centrifuge before mak- ing photometric measurement. Treat the blank in the same manner.

URINE. Dilute 0.5 and 1 ml. of urine to 10 ml. and use 0.2 ml. of these dilutions in the manner described for the analysis of serum.

SPIXAL FLUID. Follow the procedure described for serum.

and repression of the effect of mag- nesium was 12.3 (as described in Pro- cedure).

Effect of Temperature and Serum Calcium Concentration on Color De- velopment of NFR-Calcium Complex. Increasing temperatures from 5" to 30" C. had no differential effect on the development of the NFR-calcium complex if calcium were present as calcium carbonate in aqueous solution, as naturally occurring calcium in pooled serums, or as calcium car-

EXPERIMENTAL DATA AND RESULTS

Determination of Optimum Con- centration of NFR. Baar (1) did not determine the optimum concentra- tion of K F R to obtain maximum color development with serum or calcium standards. He prepared a X F R working solution by diluting 8 ml. of a 100 mg. yo K F R stock solu- tion to 50 ml. and used 2 nil. of this dilution n i t h each 0.1 ml. of serum, which was equivalent to 0.32 nig. of S F R . I n our method 10 nil. of a 100 mg. % solution of N F R was diluted to 50 ml. and 3 ml. of the diluted solution was used for each 0.1 ml. of serum, or 0.6 mg. of N F R , n-hich is about 100% more than tha t used by Baar (1). Table I indicates tha t ap- proximately 0.6 mg. of N F R is the op- timum concentration to use for each 0.1 ml. of serum.

Determination of Optimum Sodium Hydroxide Concentration for Maxi- mum Color Development and Maxi- mum Repression of Effect of Magne- sium on Color Development of NFR- Calcium Complex. Baar ( I ) stated tha t "optimum conditions appear to be a t or above p H 12.60," to obtain maximum absorbances in the binding of calcium with N F R . Baar prepared dilute nuclear fast red reagent in 0.1N SaOH. We obtained optimum color de- velopment by preparing N F R in 0.05N NaOH for sera containing normal amounts of magnesium. However, in the presence of abnormal amounts of magnesium (5 mg. %), 0.05N failed to repress the effect of magnesium, and 0.075X sodium hydroxide or higher was required (Table 11). Above 0.0750N sodium hydroxide, some intensity of color development of N F R was lost. Baar ( I ) did not demonstrate repression of the effect of abnormal amounts of magnesium. He added 2 mg. % mag- nesium to his dilute N F R reagent, which gave no correction for the presence of abnormal amounts of magnesium. We have observed that magnesium could be added to serum up to a value of 5.2 mg. % without any increase of color intensity of the nuclear red dye, as shown in Table 111. The p H of our final mixture of N F R and sodium hy- droxide for maximum color development

Table I. Determination of Optimum Concentration of Nuclear Fast Red in

Stock Solution At 20' C.

(All conditions, except dye concentration, same as described in Procedure)

Concn. of Dye in Stock

S~ lu t ion ,~ Dye, Mg./ Pooled 10 Mg. hIg./lOO 0.1 M1. Serum, % Ca

M1. Serum % T Std.,%T

25 0.15 66 64 50 0.30 56 54 75 0.45 54 52

100 0.60 52 51 125 0.75 58 57

5 No differences noted in purified (NFR) obtained from different sources noted under reagents.

Table II. Optimum Concentration of Sodium Hydroxide for Maximum Re- pression of the Effect of Magnesium on

Color Development

(All conditions, except NaOH concentra- tion, same as described in Procedure)

Pooled Serum, yo T 1.3 mg. 4 mg.

Concn. of Mg/100 Mg/100 NaOH, N ml. ml.

0,0250 50 41 0.0375 44 36 0.0500 43 40 0 I 0625 43 41 0.0750 45 45 0.1000 48 48 0.1250 53 53

Table 111. Effect of the Addition of Increasing Amounts of Magnesium to a

Serum Containing 1.2 Mg. 70 Magnesium

% 70 Mg Trans- Mg Trans-

Added mit- Added, mit- Mg. yo tancea Mg. yo tance"

0 48 6 44 1 48 7 43 2 48 8 42 3 48 9 41 4 48 10 40 5 46

6 Coleman Spectrophotometer, Jr., No. 6.

VOL. 33, NO. 4, APRIL 1961 0 553

Figure 1. Effect of temperature on color development of nuclear fast red complex

8 x 0 albumin fraction V

10 mg. % calcium standard IO mg. % pooled serum 1 0 mg. % calcium standard in 7% bovine

bonate added to 7% bovine albumin fraction V (Figure 1). At higher tem- peraturcs slightly lcw color developed. This diffcrence was fairly uniform re- gardless of the calciuni concentration, as the slopm of the absorbance curves obtained a t differcrit temperatures with sera containing different amounts of calcium (6.6 to 15.8 mg. %) were of the same magnitude (Figure 2).

Opt imum Spectrum for Measure- ment of Light Transmittance of NFR- Calcium Complex. Light absorption of the XFR-calcium complex was studied n i th different spectropho- tometers. Considerable differences in light transmittance through solutions of the KFR-calcium complex nere found rvith different spectrophotom- eters. Absorption light was maxi- mum a t 575 mp for the Coleman, Jr., S o . 6, a t 590 mp for the Beckman DU (S.W. 0.01 mp), a t 575 mp for the Spectroriic 20 (Bausch B: Lomb) and a t 560 for the Klett photoelectric color- imeter (Figure 3). An unusuallystrong absorbance mas obtained with the Spectronic 20, as only 0.02 ml. of serum was rcquired Cvrept for the Klett, all measurements were made a t 5-mp intervals.

Standardization Curves Obtained with NFR-Calcium Method. Stand- ardization curves obtained with 10

mg. yo calcium in aqueous solution, with 10 mg. yo calcium in 7Yo bovine albumin, and n i th 10 mg. % calcium and 4 mg. magnesium per 100 ml. in 7% bovine albumin (Figure 4) were identical. Transmittance read- ings n ere obtained by reading against a blank, to which no magnesium mas added. Eaar piepared standardiza- tion curvcs by reading against KFR reagent blanks containing 2 mg. 70 magnesium.

Stability of NFR Reagent. Stock NFR reagent xias stored a t room temperature and refrigerated in Corn-

5 IO I5 20 25 37 TEMPERATURE C"

Figure 2. Effect of serum calcium level and temperature on color develop- ment of nuclear fast red complex 1. 2. 3. 4.

Serum calcium, 6.6 mg. per 100 ml. Calcium standard, 10.0 mg. per 1 0 0 ml. Serum calcium, 12.2 mg. per 100 ml. Serum calcium, 15.8 mg. per 100 ml.

WAVE LENGTH [my)

Figure 3. Spectral transmittance curves obtained on different photometers with nuclear fast red-calcium method as described in procedure

1. Coleman, Jr., spectrophotometer No. 6, 0.2 ml. of standard 2. Beckman DU spectrophotometer, 0.2 ml. of standard 3. Spectral 20 (Bausch & tomb) spectrophotometer, 0.02 ml. of standard 4. Kleft photoelectric colorimeter, 0.2 ml. of standard. All standardization curves prepared with 1 0 mg. 70 calcium standard in 7% bovine albumin.

ing borosilicate glass, polyethylene, and amber bottles for as long as 70 days (Table IV), without any loss of activity or chromogenic properties in the formation of the KFR-calcium complex.

Recovery of Added Calcium. Good recoveries of calcium n ere obtained with triplicate determinations of sera before and after the addition of 1 to 15 mg. % calcium (Table V).

'Table IV. Stability of Stock NFR Reagent as Measured with Pooled Serum and Calcium Standard

Days 1 3 7 21 3 5 56 70

Per Cent T

1. Std. 49 49 49 49 49 50 50 1. Serum 49 49 49 50 49 51 51 2. Std. 49 49 49 49 50 51 50 2. Serum 49 49 49 49 51 51 51 3. Std. 49 40 49 49 49 50 EO 3. Serum 49 4 9 50 50 50 51 0 I 4. Std. 49 49 49 49 49 50 50 4. Serum 49 49 50 49 50 51 51 5 . Std. 49 49 49 50 50 50 50 5 . Serum 49 49 49 50 51 51 51

1. 2. 3. 4. 5.

Refrigerated in borosilicate bottle at 4" C. Room temperature in borosilicate bottle. Stored in dark glass bottle at room temperature. 0.02y0 Sterox added and stored at room temperature in borosilicate bottle. Room temperature in polyethylene bottle.

-

Table V. Recovery of Calcium Added to Serum as Measured with NFR-

Calcium Method Calcium Added, Found, Recovered, Mg. % Mg. % % 0.0 9 . 2 . . .

9 . 2 . . . 9.2 . . .

1 . o 10.2 100,o 10.2 100.0

2.0 11.3 105.00 11.2 100.00 11.2 100.00

3.0 12.2 100.00 12.0 93.5 12.1 96.5

Effect of Serum Lipides. Calcium detprminations were carried out on a series of sera (Table VI), which con- tained abnormal amounts of lipides, 0.88 to 4.8%, by the direct flame photometric and the S F R colorimetric methods. Good agreement of calcium determinations was obtained with these methods. It has been shown previously (8) that the presence of ab-

554 * ANALYTICAL CHEMISTRY

Figure 4. Standardization curves ob- tained with nuclear fast red-calcium method as described in procedure

0 0 Calcium standard, diluted in 7% bovine albumin fraction V X Calcium standard, diluted in 7% bovine albumin fraction V containing 4 mg. % mag- nesium

Calcium standard, 10 mg. %

Table VI. Effect of Excess Serum Lipides on NFR Color Development

Ca, Mg./100 M1. Direct flame (8)"

spectro- photom- NFR Lipides,

Total

N O . eter method % 1 9.7 10.0 1.230 2 9 .7 10.0 3.040 3 10.6 10.8 1.100 4 9 . 4 9 . 6 1.650 5 9 . 4 9 . 2 2.300 6 9 . 4 9 . 2 2.175 7 9 . 5 10.0 2 000 8 10.6 io.8 0.800 9b 9 . 8 9 . 8 4.800

10 10.6 10.6 0.900 11 10.0 10.0 1.800 12c 52.0 50.0 3.300 13* 10 .4 10.4 4.550 14b 9 . 2 9 5 4.100

Beckman flame photometer with photomultiplier attached.

Milk and all sera containing 4% or more lipides and their blanks were shaken with 3 ml. ether for 5 seconds, then cen- trifuged for 10 minutes a t 3000 r.p.m. and calcium determined as directed in Pro- cedure.

Sample of milk diluted 1/10, for final color development.

normal amounts of lipides do not inter- fere in the determination of serum ca!cium by direct flame photometric measurement. Milk containing 3.3 % lipides and 50 to 62 mg. of calcium per 100 ml., if diluted to 1 to 10, could be analyzed for calcium by the KFR method.

Effect of Phosphorus. The addition of 10 to 500 mg. of phosphorus per 100 ml. of phosphoric acid to urine (Table VII) had no effect on the de- termination of calcium by the NFR, direct flame photometric (8) , and Elliott-Pearson (3) methods, as all of these methods were in good agreement.

Effect of Proteins. Calcium was determined in sera containing different amounts of serum total proteins (4.9 to 9.1 gram yo) by the KFR and direct flame photometric methods; the re-

sults were in good agreement (Table

Several established calcium methods, VIII). Table VIII. Effect of Different Amounts

of Serum Proteins on Calcium Determination

Table VII. Effect of Increasing Amounts of Phosphorus in Urine on Determina- tion of Calcium by the NFR Method

Ca, Mg./100 M1. Direct

Phos- flame phorus photo- Elliott-

Added," metric Pearson Mg. % NFR (8 ) ( 3 )

0 10.6 10.4 10.6 10 10.6 10.6 10.4 30 10.6 10.4 10 .5 50 10.6 10.6 10.4 75 10.6 10.5 10.6

100 10.6 10.6 10.6 150 10.6 10.6 10 .6 250 10.6 10.4 10 .8 300 10.6 10.6 10.7 500 10.6 10.6 10.6

a Added as HIP04 to a urine containing 55.4 mg. phosphorus per 100 ml.

Ca, Mg./lOO iM1. Direct

Total flame Protein, photo- Gm./100 metric

No. M1. NFR (8 ) 1 9 . 1 10.0 10.0 2 8 . 7 10.0 10.0 3 8 . 2 10.8 10.6 4 8 . 5 9 . 8 10.0 5 5 . 4 7 . 8 8 . 0

Corinth dye (7), direct flame photo- metric (8), and Elliott-Pearson (3) (oxalate precipitation and photometric measurement of excess permanganate) were compared t o the NFR method as described in this report for the de- termination of calcium in serum, urine, and spinal fluids and were found to be in excellent agreement (Table IX).

Table IX. Comparison of NFR Method with Other Methods for the Determination of Calcium

Serum No.

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20

21 22 23 24 25 26 27

28 29 30 31 32 33

Corinth Dye ( 7 ) , Mg. yo

9 . 4 9 . 0 9 . 5 9 . 2 9 . 0 9 . 2 8 . 3 9 . 5 9 2 9 . 0 9 . 9 9 . 9 8 . 9

10.6 10.2 10 .2 8 . 2 9 . 7 7 . 9

10.0

107 76 60 48

102 24 64

5 . 4 5.1 5 . 0 5 . 6 4 . 6 4 . 8

NFR, Mg. %

9 . 2 8.8 9 . 4 9 . 2 8 . 9 9 . 2 8 . 4 9 . 4 9 . 2 9 . 0 9 . 6 9 .6 9 . 0

10 .6 10.0 10.0 8 . 2 9 . 8 7 . 8 9 . 8

110 79 61 50

103 22 63

5 .4 5 . 0 4 . 8 5 . 6 4.4 4 . 8

Coleman Flame (8) 31g. yp Rlg. Y0b

Serum '3.4 9 . 1 9 . 0 8 . 8 9 . 7 9 . 5 9 . 5 9 . 4 9 . 2 8 . 9 9 2 9 0 8 . 6 8 . 4 9 6 9 . 4 9 . 4 9 . 1 9 . 2 9 . 0 9 . 6 9 . 6 9 6 9 . 7 9 . 2 9 2

10 .4 10.3 9 . 8 10.0 9 . 9 9 . 9 8 . 3 8 .0 9 . 8 9 . 5 8 . 0 7 . 8

10 .0 9 . 7

Urine . . . 108 . . . 75 . . . 60 . . . 49 . . . 103 . . . 21

62

CSF 5 . 5 5 . 2 5 . 3 5 . 0 5 . 0 4 . 8 5 . 8 5 . 6 4 . 6 4 . 4 5.1 4 . 9

Direct method. Calcium first separated from serum a8 oxalate.

Beckman Flame (8) Mg. Yo" Mg. ?ob

9 2 8 .8 9 . 4 9.2 9 . 0 9 . 0 8 . 4 9.6 9 . 2 9 0 9 . 6 9 6 9 . 2

10.4 10 0 9 . 9 8 . 0 9 .6 7 . 8 9 8

110 77 62 51

105 22 65

5 . 2 5 . 0 4 . 8 5 . 9 4 . 7 5 . 0

9 . 1 8 . 8 9 .4 9 . 2 9 . 0 9 . 0 8 . 4 9 . 5 9 . 2 9 . 0 9 . 7 9 . 7 9 . 0

10 .4 10.0 10.0 8 . 0 9 . 5 8 . 0 9 . 8

107 76 63 50

102 21 62

5 . 3 5 . 0 4 . 7 5 . 6 4 . 4 5 . 0

Elliott- Pearson (31,

h k . 70

9 . 3 8 . 7 9 . 4 9 . 3 9 .0 9 . 0 8 . 3 9 . 4 9 . 2 8 . 9 9 . 8 9 . 6 8 . 9

10 .5 10.2 10 .0 8 . 2 9.G 7 . 8 9 . 8

108 75 60 51

102 25 62

5 .0 5 . 0 4 . 4 5.6 4 . 4 5 . 0

VOL. 33, NO. 4, APRIL 1961 555

The Coleman flame photometer was concentrations of bilirubin and greater not satisfactory for urinary calcium de- hemolysis blanks could not be used terminations. for making satisfactory corrections. ( 5 ) Ihid. , pp. 689-93.

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- (2) Chilcote, M. E., Wasson, R. D., C h . calcium color complex. At higher (3) Elliott, J. E., Pearson, P. B., J . Lab. Accepted January 5, 1961.

Clin. Med. 31, 1262-6 (1946).

( 4 ~ ~ ~ ~ ~ a t ~ ; l , ti, ~ o ~ 1 7 $ l ~ a , , ” ” * (6) Kingsley, G. R., Robnett, O., Ibid.,

(1) Baar, s.. Clin. Chim. Acta 2, 567-75 (8) Kingsley, G. R., Schaffert, R. R., (1957).

Chem. 4, 200-10 (1958). RECEIVED for review August 15, 1960.

LITERATURE CITED 27, 223-30 (1957). (7) Ihid., 29, 171-5 (1958).

API’AL. CHEM. 25, 1738-41 (1953).

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 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 i s only 0.1 to 1 ml.

OLLOWING the development by the F 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 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 that used in the earlier work (8, 6). The source was a twin-electrode calcium/ 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-

tifier, and meter are made commercially by Techtron Appliances, South Mel- bourne, 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 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 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 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-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 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 oren-dried calcium car- bonate in the minimum quantity of hydrochloric acid and diluting to vol- ume. Standard niagnesium 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

Two halves are dowelled and screwed together. Mear- urements are in rnrn.

556 ANALYTICAL CHEMISTRY