Polarographic Analyses of Mixtures of Predaisoae and Cortisone PETER KABASAKALIAN, SAM DELORENZO,and JAMES MCGLOTTEN Research Division, Schering Corp., Bloom field,
N. 1.
A polarographic procedure has been developed for t h e analysis of prednisone-cortisone m i x t u r e s w i t h an accuracy w i t h i n 4%. A n additional procedure for use w i t h m i x t u r e s r i c h (90qo or over) in one c o m p o n e n t gives results good t o 170.
THE
possibility of using the polarograph as an analytical tool in the analysis of A4-3-ketosteroids was first pointed out by Eisenbrand and Picher (1). These authors examined testosterone, progesterone, and deoxj-corticosterone in 90% ethanol solutions 0.LV in lithium chloride, and found a linear relationship bctxeen concentration and wave height for the compounds. Shortly thereafter, Wolfe, Hershberg, and Fieser (7') made a very comprehensive study of the polarographic analyses of ketosteroids. h great portion of the work done by these authors concerned the determination of the Girard T derivatives of the saturated and unsaturated ketosteroids, although they examined srveral of the free A4-3-keto compounds in alkaline 2-propanol solutions and their findings substantiated those of Eisenbrand m d Picher. Since that time a number of articles have been published convcrning the polarographic assay of A4-3-ketosteroids, Hershberg, Fl-olfe, and Fieser (2) developed an assay method for 3-hydroxyA5-steroids based on the polarographic determination of the corresponding 3-keto-A4-compounds after oxidation with aluminum tert-butoxide. Morris and Williams (4)and Morris (3) combined chromatography and polarography in developing a procedure for the determination of blood corticoids. Zuman, Tenygl, and Brezina (8) proposed a system for determining total steroid content of a mixture containing testosterone, methyltestosterone, progesterone, cholestenone, and deoxycorticosterone. They al-o suggested conditions under which deoxycorticosterone could be determined in the presence of testosterone and progesterone i n the presence of methyltestosterone. The present work describes a polarographic method capable of assaying mixtures of a A4-3-ketosteroid (cortisone) and it A1i4-3-ketosteroid (prednisone). This assay is very useful for following the microbiological transformation of cortisone to prednisone (5, 6).
CHzOH
c =o
I
$HzOH
- --OH
"&CORTISONE
PREDNISONE
EXPERIMENTAL
Materials. The steroid compounds were made and purified in the Schering Laboratories. All chemicals used in the polarographic solutions were of reagent quality. Apparatus. All polarographic measurements !\-ere made with the Sargent Model XXI recording polarograph. The polarographic cell was a small H-cell (3-ml. sample volume) with an anode compartment consisting of a normal calomel electrode. The capillary constant, m2I3t1l6, was 1.80 mg.2/3sec.-1/2 a t an
open circuit with the electrode immersed in a 0.1N potassium chloride solution. Buffer Solution. The stock buffer solution was prepared by dissolving 1.0 mole of anhvdrous sodium acetate in 1.OM acetic acid in al.0-liter volumefric flask and then diluting to volume with more 1.O.V acetic acid. Basis for Procedure. Both prednisone and cortisone give well-formed, reproducible polarographic waves (Figure 1) in a 50% methanol solution buffered a t p H 5.5 with sodium acetateacetic acid buffer. The position of the waves (E112 = -1.20 and - 1.36 volts for prednisone and cortisone, respectively) is independent of concentration and the wave heights are linearly dependent on concentration over fairly wide concentration ranges. Because of the wave separation it seemed likely that mixtures of the compounds might be determined. However, on examining the individual polarograms it was evident that the wave of one compound in a mixture ~ o u l dencroach on the wave of the other. To correct for this overlapping, equations were set up that \Todd account for the current of a mixture a t each of two voltage points corresponding to voltages near the platcau of each compound.
+ Y 1 g = il k?X + k'ny = i?
k1X
(1)
(2)
where k and k' are the current constants ( i d / C ) for prednisone and cortisone, respectively. The subscripts 1 and 2 refer to the two voltage points, - 1.31 volts (prednisone plateau), and - 1.47 volts (cortisone plateau). The follon-ing values for x and y, the concentration of prednisone and cortisone, respectively, are ohtained: (3)
(4) The use of these equations should yield fairly accurate results in spite of small amounts of wave encroachment. The results of further experimentation showed this to be so. General Procedure. All polarographic sample solutions were made by neighing the desired amount of steroid into a 10-ml. volumetric flask on a microbalance. This material was dissolved in 5 ml. of methanol, 1 nil. of buffer was added, and the solution was diluted to volume with viater. T a o portions of the sample solution n-ere used to rinse the cell and a third portion was deaerated 13 ith nitrogen for 10 minutes before polarographing. All current measurements 1% ere made by the point increment method with observations at -0.90 (zero point), -1.31 (prednisone flateau), and -1.47 (Corti-one plateau) volts us. N.C.E. Samp e currents Tvere corrected for a blank solution measured a t the same voltages The temperature was maintained a t 25" i0.2" c. Determination of Prednisone-Cortisone Mixtures. Several samples of prednisone and cortisone (0 5 mg. per ml.) were polarographed in the above manner and current constants obtained for each compound a t each of the t n o plateau points. A number of synthetic mixtures TTere then made by IT eighing a small amount of prednisone and cortisone into the same flask in such quantities that the total steroid content of the final solution was approximately 1 mg. per ml. These mixtures were examined a t the three designated voltage points and the prednisone and cortisone concentrations determined by substitution of the currents obtained into the equations derived earlier. The experimental results are compiled in Table I.
1669
ANALYTICAL CHEMISTRY
1670 Determination of Mixtures High in One Component. It was observed experimentally that the current constants for both prednisone and cortisone a t lon concentrations R-ere substantially higher than those used in the above work. iZny change in the actual current-concentration relationship from that used to set up the equations would, of course, lead to errors in the determination. Fortunately, the deviations from linearity for each compound occur at low solute concentrations, and current error introduced by the higher current per unit concentration value of the steroids in this region is small relative to the total current involved. For this reason the maximum absolute error introduced in a determination is from 3 to 4% of the major component. However, Kith material rich in one of the components, the method can be made to yield more accurate results. For this work solutions of 1.0 and 0.1 mg. per ml. of both cortisone and prednisone were run and current constants determined for the compounds a t the appropriate voltages. One series of synthetic mixtures was made up containing 90 to 100% prednisone ;nid another series was made up containing 90 to 100% cortisone. These mixtures were then assayed using the special current constants obtained above (Table 11). For the mixtures rich 111 prednisone the constants obtained for the concentrated predni-one solution and the dilute cortisone solution were used. For the mixtures rich in cortisone the opposite constrtnts mere used.
A.
Table I. Prednisone Concn., hlg./Ml. 1.005 0.937 0.815 0,740 0.613 0 503 0 306 0.213 0.105 0.000
Analysis of Prednisone-Cortisone Mixtures Cortisone Concn., XIg./JIl. 0 000 0 105 0 206 0 307 0 408 0 503 0 761 0 805 0 919 1 009
A Prednisone Found Calcd. 96.8 100.0 86 8 78 8 68.7 58 2 51 6 29.0 21.3 10.9 -0.6
89.9 79.8 70.7 60.0 50.0 28.7 20.9 10.3 0.0
yo Cortisone Found
Calcd.
-0.7 11.0 20.0 30.2 41.1 49.3 70.6 78.7 86.0 96.0
0.0 10.1 20.2 29.3 40.0 50.0 71.3 79.1 89.7 100.0
Current constants obtained from samples containing 0.5 mg. per ml. of prednisone and cortisone.
Table 11. -4nalysis of Prednisone-Cortisone IZixtures Piednisone Concn., Mg./JIl. 1,005 1.217 1.101 1.005 0 908
Cortisone Prednisone, % Cortisone, % Concn., JIg./.\Il. Found Calcd. Found Calcd. 1 0 M g . / l l l , Prednisone, 0.1 hfg./hfl. Cortisone 100.8 100.0 -0.8 0.0 0,000 0.040 0 060 0.100 0.100
95.9 94.7 91.0 89.9
96.8 94.8 91.0 90.1
2.1 4,0 8.1 8.8
3.2 5.2 9.0 9.9
0.1 l\lg./Ml. Prednisone, 1.0 hIg./hfl. Cortisone 0.000 0.020 0.037 0.080 0.100
PREDNISONE
1.009 1.009 1.014 1,124 1.018
-1 0 2.6
3.5 5.9 8.5
0.0 1.9 3.8 6.6 8.9
100.0 97.2 96.5 92.4 90.7
100.0 98.1 96.2 93.4 91.1
B. C O R T I S O N E
I .
MIXTURE
Table 111. Precision of Method
I ua.
Sample Concn., hlg./Jll. 0 520 0.510
1,
< IZ
0 3 0
/ / 1.1
%
L r.
Cortisone,
%
86.9 87.5 87.1 87.3 87.7 87.7 88.4 89.6 88.1
11.7 12.7 13.5 12.7 13.1 12.3 12.4 12.0 11.9
Mean 87.8 Std. dev. 1 0 . 8
12.4 h0.6
0,502 0.536 0.504 0 505 0.517 0 501 0.537
W
(L
A
Prednisone,
J
1.3
1.5
POTENTIAL, volts
Figure 1. Polarograms of prednisone, cortisone, and a mixture of the two compounds in 50% methanol at pH 5.5
component and 2% in the minor component. ;\lore accurate results can be obtained for the mixtures rich in one component by the application of special current constants. A prednisonecortisone mixture can be assayed with a precision within ~ t 0 . 8 7 ~ (1 sigma). LITERATURE CITED
Eisenbrand, J., Picher, H., 2. physiol. Chem. 260, 83 (1939). (2) Hershberg, E. B., Wolfe, J. K., Fieser, L. F., J . Am. Chem. Soc. (1)
62, 3516 (1940).
Precision. -4synthetic mixture of prednisone and cortisone was dissolved in methanol and precipitated, then dried, and ground to a powder. Ten separate samples of this mixture were weighed and the components determined (Table 111). ANALYSIS OF RESULTS
The experimental results indicate that both components of a prednisone-cortisone mixture can be determined polarographically. If the standard current constants are determined from solutions whose steroid concentration is one half that of the sample solutions assayed, the prednisone-cortisone content can be determined with a maximum error of 4% absolute in the major
(3) hforris, C. J. 0. R., Rec. trap. chim. 74,476 (1955). (4) Morris, C. J. 0. R., Williams, D. C., Biochem. J . 54, 470 (1953). (5) Nobile, A., Charney, W., Perlman, P. L., Herzog, H. L., Payne, C. C., Tully, 3f. E., Jevnik, M. A4., Hershberg, E. B., J . Am. Chem. Soc. 77, 4184 (1955). (6) Stoudt, T. H., hIcAleer, W. J., Chemerda, J. AI., Kozlowski, 31. A., Hirschmann, R. F., Marlatt, V., Miller, R., Arch. Biochem. and Biophys. 59,304 (1955). (7) Wolfe, J. K., Hershberg, E. B., Fieser, L. F., J . Bid. Chem. 136,653 (1940). (8)
Zuman, P., Tenygl, J., Breeina, XI., Collection Czechoslov. Chem. C o m m u n s . 19,46 (1954).
RECEIVED for review March 22, 1956. Accepted July 20, 1956. Seventh Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy. Pittsburgh, Pa., February 28, 1956.
