a n attenuation of X 1 and a 1-mv. recorder, a full scale deflection v-as obtained with 0.1 fig. of etiocholanolone. This degree of sensitivity indicates that the gas chromatographic method is potentially applicable to measurement of 17-ketosteroids in blood, where the mean values for total 17-ketosteroids are reported to be 181 pg. per 100 ml. of plasma in normal men and 126 pg. per 100 ml. of plasma in normal women (11). ACKNOWLEDGMENT
The authors gratefully acknowledge the advice of B. G. Creech, Texas Medical Center, Houston, Tes., and the technical assistance of George Roushey and Beverley hlacomber. The authors are also indebted to Leon Schwartz for photographing the illustrations. LITERATURE CITED
(1) Bradlow, H. L., Gallagher, T. F., J . Biol. Cheni. 229, 505 (1957). (2) Burstein, S., Lieberman, S., Ibid., 233, 331 (19,58). (3) Burton, R. B., Zaffaroni, A,, Keutmann, E. H., Ibid., 188,763 (1951). (4) Bush, I. E., “Chromatography of \ - - - - ,
Steroids,” Pergainon Press, Oxford, 1961. (5jCooper, J. A., Creech, B. G., Anal. Biochem. 2, 502 (1961). (6) De Paoli, J. C., Nishizawa, E., EikNes. K. B., J . Clzn. Endocrinol. Metab. 23, 81 (1963). ( 7 ) Dobriner, K., J . Clin. Invest. 32, 940 I1 9.53). \----,
(8) Garrett, H. E., Creech, B. G., Horning, E. C., DeBaker, M. E., Cardiova&ular Research C a t e r Bull. (Texas
hledical Center, Houston, Tex.) 1, 15 (1962). (9) Haahti, E. 0 . h., Vanden Heuvel, IT. J. A., Homing, E. C., 9 n a l . Biochem. 2, 182 (1961). [ l o ) Hartman, I. S., Wotiz, H. H., Steroids 1 , 33 (1963). (11) Hudson, B., Oertel, G. W.,Anal. Biochem. 2, 248 (1061). (12) Jacobsohn, Q. M.,Lieberman, S., J . Riol. Chem. 237, 1469 (1962). (13) Kappas, A . , Gallagher, T. F., J . Clin.Invest. 34, 1566 (1955). (14) Kirschner, ?*I. A., Lipsett, X I . B., J . Clin. Endocrinol. Jietab. 23, 255 (1963). (15) Lieberman, S., Hariton, L. B., Fukushima, D. IC., J . Anr. Chenz. Soc. 70, 1427 (1948). (16) Lieberman, S., Mond,. B., Smyles, E., “Recent Progress in Hormone Research,” (i. Pincus, ed., p. 113, Academic Press, New York, 1954. (17) Lipsky, S. R., Landowne, R. A , , ANAL.CHEM.33, 818 (1961).
(17) Peterson,
R. E., Pierce, C. E., Lipids and Steroid Hormones in Clinical Medicine,” F. W. Sunderman, F. W. Sunderman, Jr., eds., p. 158, J. B. Lippincott Co., Philadelphia, 1960. (19) Purnell, J. H., Ann. A-. Y.Acad. Sci. 72, 592 (1959). (20) Romanoff, L. P., Rodriguez, R. M., Peelye, J. &I., Parent, C., Pincus, G., J . Clan. Endocranol. X e t a b . 18, 1285 (1958). (21) Salamon. I. I., Dobriner, K., J . Biol. Chem. 204, 487 (1953). (22) Sparagana, LI., Mason, Vi’. B., Keutmann, E. H., ANAL. CHEM.34, 1157 (1962). (23) Talbot, K. B., Berman, R. A., MacLachlan. E. A., J . Biol. Chem. 143, 21 1 (1942). Vanden Heuvel, W. J. A., Creech, G., Homing, E. C., Ann. Biochem. 191 (7962). Vanden Heuvel, W.J. A., Homing, C., Biochem. Biophys. Res. Commun. 3, 356 (1960). 126) Vestkreaard. P.. Claussen. B.. ilcta ‘ Endocrina. Suppl. ’64, 35 (1962). ‘ (27) Wheeler, 0. H., Chem. Reas. 62, 205 (1962). RECEIYEDfor review April 8, 1963. Accepted June 14, 1963. Investigation supported hy P. H. S. research grant CA-01003-12 from the National Cancer Institute and P. H. S. Research Facilities Grant 8M-01-Fr-44-03.
Fluorometric Determination of Calcium in Blood Serum BARBARA L. KEPNER and DAVID M. HERCULES Department of Chemistry, Juniafa College, Huntingdon, Pa.
b A-fluorometric method for the determination of calcium in blood serum is described. The method is based on the formation of a fluorescent chelate between calcium and Calcein in a strongly alkaline solution. N o interference from magnesium ion or from protein was encountered. The variables studied included the effect of pH, Calcein concentration, and time of standing. Agreement with clinical analysis of serum samples was
*I%.
D
v : ~ m m ~PIOX . \ o b ’ WP: cdciuni colitent of blood scruni has been acromplished by several methods. The method of Clark and Collip ( 1 ) involved precipitation of calcium directly from blood serum a5 the oxalate and subsequent t i t r a t i u with permanganate. A colorimetric method was devised by Rowe and Kahn utilizing precipitation of tricalcium phosphate and subsequent colorimetric determination of the phosphorus (4). Flame photometry is widely used for the determination of calcium in blood serum, as is titration of the calcium with EDTA using one of a 1238
ANALYTICAL CHEMISTRY
variety of indicators to estimate the equivalence point (6). Calcein and similar compounds (2, 5 ) are senqitive fluorometric reagents for calcium, and Calcein was used as the reagent for the method described in this communication. This method is precise, rapid, and requires only microliter quantities of blood serum. Some difficulties were encountered owing to the instability of Calcein in aqueous solution and to a fluorescent impurity3 probably traces of calcium present in onc ( i f the reagents. Hoivever, by careful l)ipetting, the method showed a rchkive standard deviation of f1 % and cornparable agreement with Serum analy-eh performed at the Altoona. Pa. Hospital and with Clinical Control Lerum Standards. EXPERIMENTAL
Reagents and Solutions. The water used t o prepare all qolutions wa? distilled in a Barnstead still and then passed through a n ion eschangp column. A11 solutions werp &xed in polyethylenc bottlcq. Rengcnt gr:idr chcmicals I\ (’re u w 1 throughout.
Standard ralcium solution: A 001ution containing 40.0 mg. of calcium per liter was prepared by dissolving calcium carbonate in A minimal Quantity of HCl and diluting to 1 liter. Calcein qolution: Eit,her Calcein (G. Frederick Smith Chemical Co. fluorescein iminodiacetic acid) or Calcein W (G. Frederick Smith Chemical Co. fluorescein iminodiacetic acid disodium d t ) was dissolved in fluorescencegrade propylene glycol (Hartman-Leddon Co.. Philadelphia. Pa.) to preparr a solution containing 60 mg. of Calcein I)er liter. For :tqucoui iolution- of Calrcin. thc dye u-a. d i 4 v c d i i i A minimal aniount of 0.10s KOH sntl diluted to volume. It nas neceqsary to add a m a l l amount of EDTA to the Calcein solution to permit balancing of the blank on the fliioromctei. The amount of EDTA I eyuiird e5tabliahed by titration and n as iouiid to be constant lor Calcein solutions prepared from the same bottle of solid reagent. (A typical amount of EDTA added was 1.00 ml. of 0.030Jf EDTA per 100 ml. of Calcein solution.) However, variation in the smount of EDTA requiied w a i encountel cd n hen Calcein solutions 71 ere prepared from diflcrcnt lmttlcs of thc >olid rcngcnt.
An independent laboratory checking the present method ( 3 ) has found the addition of E D T A to the Calcein reagent to be unnecessary, even Tvhen using a dye sample which r q u i r e d 1.03 nil. of 0.03056 EDTA per 130 ml. of Calcein solution u h e n used in our laboratory. This indicates that the calcium contamination arose main1 \* from the KOH used or from traces of cstlcium remaining in the purified water. Apparatus. T h e instrument used was a G. K. Turner A&iociates(Palo Alto, Calif.) Model ' 10 fluorometer equipped n-ith a S o . 7-60 (360 mp) primary filter, a Xo. 8 (485-mp cutoff) secondary filter and a S o . 110-850 ultraviolet lamp (near UV). This instrument was quite sensitive and showed good long-term and sho %-term stability. The filter combination used was selected to provide opt mum conditions from those combinal ions of filters ayailable with the Turner fluorometer. The chosen combination is reasonable in terms of the spectral properties of (6). the Calcein-calcium chelate Phillips ( 3 ) has indicated recently t h a t excitation a t 436 mp is also possible using the Turner 2-4 plus 47B primary filter combination and the 2-4-12 secondary filter n ith appropriate neutral filters. Procedure. T h e recommended procedure for t h e determination of t h e calcium content of blood serum with Calcein is: 5.00 nil. of 2 . 0 0 5 KOH were added t o each of two 25-ml. volumetric flasks; 20 pl. of blood serum were pipetted into the sample flask; 1.0 ml. of Calcein W solution was added to each fla>k; reagent: Jvere mixed b y swirling and diluted tr, volume. The Turner fluorometer wa; balanced ivith the blank and the sam71e read relative to the blank. The milligrams of calcium per 100 ml. of blood serum were calculated from a calibrstion curve. The procedure utilized in thoqe studiei involving n-atcr as a solvent for Calcein was the samr except that 5.00 ml. of 0.40S KOH m r e used. RESULTS
Table I s h o w typical calibration data obtained using thc procaedure described above, but substitutin,; standard calcium solutions for blood samples. Individual points on the calibration curve could be reproduced froin day to day within cxpcrimcntal error (iI yo) whrn the same Cwlccin solution was used, but drviations of 1 4 % ivcrc encountered when different Calcein solutions mere used. Therefore when analyzing blood samples a complete calibration curve was p r e p a r d for each batch of Calceiii solution, alth ,ugh for clinical use such a procedure should not be necessary. It should he noted t,hat each sample contained 0.1 pniole of Calcein reagent, per 25 nil. and between 0 and 0.07 pinole of ralcium in th? same volume. Therefore, the calibration curve begins to divcrge from linearity as the mole ratio
~
of Calcein and calcium approach s 1 : l . This indicates the procedure as outlined is not useful for a wide range of calcium concentrations. However, this presents no problem for the determination of calcium in blood serum since calcium concentrations in blood lie within a narrow range, and fall between the fluorometer dial readings of 25 and 50, which is within the linear portion of the calibration curve. When serum samples a e r e analyzed fluorometrically, the analyses presented in Table I1 were obtained. From these data i t is evident t h a t the fluorometric results agreed with clinical laboratory results and standard serum controls to within .tly0. T h e agreement between the analyses reported in Table I1 and those of the clinical laboratory is amazingly good. It should be remembered that both sets of analyses represent the average of several results and that they were obtained under ideal conditions. I n addition to calcium, blood serum contains magnesium and protein, both of which constitute possible interferences with the present method. Because these constituents vary in concentration in serum, a n investigation of their effect on t h e fluorescence of the calcium-Calcein system was conducted. A 100-fold excess of magnesium ion did not affect the calcium analysis of simulated serum samples. Likewise, adding protein (globulin and albumin) in two-fold excess of the concentrations found in blood produced no change in the calcium analysis of simulated serum samples. From these studies i t can be concluded t h a t magnesium ion and protein are not interferenceq for the mpthod. Calrein IT Tvas readily soluble in water, but it. solutions underwent rapid decomposition when exposed to light. Even in the dark, wlutions of Calcein ]Ir deteriorated so rapidly that it was necessary t o construct a new calibration curve daily to compenwte for decompoyition. A series of qualitative studies indicated that hydrolyqis of the Calcein 77' 1%-asprobably the major problem. Therefore a nonaqueous solvent 1v35 qought in which Calcein W nould he stablp. Calcein JT I\W wluhle in l)rnpylw(, g l col. ~ :inrl +aliitioni of t h r dye w r e stable in this wlveiit for at least tw-o \vwhs. PI pi1 when -tanding 111 sunlight. Thrwforc, the use of fluorometric-grade prop>lcne glycol to prepare the Calcein reagent was adopted, even though this constitutes wine\%hat of an incon\ enience for pipetting 1 ml. of reagent.
Table 1. Typical Calibration Data for Determination of Calcium in Blood Serum
Each sample contained 5.00 ml. of 2.005 KOH and 1.00 ml. of Calcein reagent per 25.00 ml. Ca concn. Fluorometer (rg./25 ml.1 dial reading Q 00 0.40 0.80 1.20 1.60 2.00 2.40 2.80 3.20 3.60
II.
Table
0.0 i.O 14.5 21.5 29 . 0 36.0 43.5 51 . 0 55 . 0 58 . 0 Fluorometric Analysis Blood Serum
10.4 f 0 . 1 9 . 4 f 0.05 9 . 3 f 0.1 9 . 4 f 0.05 9.3 f0.1 10.3 f 0 1 8 . 8 .t 0 0 9.9 i0.1 10.5 i0 . 1 11.5 f 0 . 1 11.5 f 0.1 i . 9 f0.1 0 . 7 f 0.05 8.8 f 0.0 9.2 f 0.1 11.7 f 0 . 2
1
2 3 4 3
6 7 8 9 10 11 12 13 14 15 16
of
9.2 10.2 S.8 10.0 10.6 11 .6 11 .6 7.8 9.8 8.8 9.lC 11.92
Fluorometric analyses n ere done using a 20-rl sample of blood serum. Data presented are for minimum of 3 deter-
minations on each sample. Precision is reported as standard deviation. Clinical results mere obtained by flame photometry at Altoona, Pa. Hospital. Data presented are average of several determinations on each sample. Hyland Labs Clinic a1 Control Serum, Normal, Lab. S o . 369Ii47 Stated value 9.1 mg./100 ml., acceptable range 8 9 to 9.3 mg./100 ml. Hyland Labs Clinical Control Serum, Abnormal, Lab. KO 3681'3 Stated value 11 9 mg./100 ml.; acceptable range 11.5 to 12.3 mg /10Q ml. Table 111. Effect of Time on Fluorescence of Blank and Sample 'l'inicb
:Ifter mixi,lg (minutes) 0 1
$4
.4i
S4 84 84 84
54 54 54 33 53 52 52
3
Blank water. a
Relative &iorescencp intensity 13lank" Sampleh $4 54
2 3
7 10 13 15 17 20
STUDY OF VARIABLES
Effect of Time of Standing. Samples showed a small apparent change in fluorescence on standing nfter mixin
