ANALYTICAL CHEMISTRY
944 -4rden, T. V., Burstall, F. H.. and Linstead, R. P., U. S. Atomic Energy Commission, D o c . CRL-AE-3 (declassified Sept. 9 , 1952).
c
Aronoff, S., Science, 110, 590 (1949). Bennett, G. I f . , and Philip, W-.G., J . Chern. Soc., 1928, 1937. Burstall, F. H., Daries, G. R., Linstead, R. P., and Wells, R.B., I b i d . , 1950, 516.
Burstall, F. H., Davies. G. R., Linstead, R. P., and Wells, R. A, S a t w e , 163, 64 (1949). Burstall, F. H., Davies, G. R., and Wells, R. A , , D i s c u s s i o n s
- Soc.. 7 . 179 (1949).
Faradau (8) (9) (10) (11)
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(12) Lederer, E., and Lederer, 31., “Chromatography,” pp 93-7, Elsevier, h-ew York, 1953. (13) I b i d . , p. 334. (14) I b i d . , p. 338. (15) Lederer, AI., iyature, 162, 776 (1918). (16) Lewis, J. d.,and Griffiths, J. 11 , A n a l y s t , 76, 388 (1951). and Elbeih, I. I. 11., J.Chem. (17) Pollard, F. H., LIcOmie, J. F. W., Soe., 1951, 466. (18) Sill, C. W., and Peterson, H. E., ANAL.Cmlr., 19, 646 (1947). (19) Strain, H. H., I b i d . , 23, 25 (1951).
\ - - - - ,
Frierson, W. J., and Jones, J. W., ANAL.CHEM., 23, 1447 (1951). Gordon, C. L., I b i d . , 26, 176 (1954). Kelley, A I . T., and Miller, H. H., I b i d . , 24, 1895 (1952). Kowkabany, G. N., and Cassidy, H. G., I b i d . , 24, 643 (1952).
RECEIVED for review d u g u s t 16, 1951. Accepted January 28, 1955. Presented in part a t the Southeastern Regional Meeting of the AJIERICAN CHEMICAL SOCIETY, Auburn, Ala., October 23 to 25, 1952.
Determination of Hydrocortisone C. R. SZALKOWSKI, M. G. O’BRIEN, and W. J. MADER Chemical Division, Merck & Co., Inc., Rahway,
N. J.
Hydrocortisone and cortisone are used extensively in pharmaceutical preparations. There is a need for a specific method to determine either hydrocortisone or cortisone. The method reported is based on the yellow color produced by hydrocortisone in a mixture of sulfuric and glacial acetic acids. This color, with a maximum absorption at 470 mp, is utilized for the identification and colorimetric determination of hydrocortisone in substance, in mixture with cortisone, and in preparations. The effects of acetic acid concentration, temperature, and time are described, and data concerning absorption maxima of other steroids and related compounds are given.
S
TEROIDS and related compounds are known to react with strong acids such as sulfuric, phosphoric, perchloric, and trichloroacetic, yielding colors or fluorescence which can be utilized for analytical purposes (3,4,12, 17). T h e reaction of steroids with sulfuric acid (5-7, 9, 10, 1.2, 1 7 ) has been used in establishing the identity of compounds isolated from biological materials. Sulfuric acid-induced fluorescence of corticosteroids has been applied to both qualitative and quantitative analysis ( 7 , 12, I S , 1 6 ) . Banes ( 1 ) treated hydrocortisone with 20% solution of concentrated sulfuric acid in glacial acetic a t 100” C. for 1 hour to obtain a series of yelloworange colors which obeyed Beer’s law a t 450 mp) the wave length of maximum absorption. Corticosterone reacted similarly, whereas cortisone and 11dehydrocorticosterone produced practically no color. Clark ( 2 ) used a mixture of 90% acetic acid and 10% sulfuric acid and a 30-minute heating to obtain similar results. Sulfuric acid and phenylhydrazine ( 1 1 ) have been used as a quantitative micromethod for 17,21-dihydroxy-20-ketosteroids, as has the colorimetric method using tetrazolium salts, 2,3,5triphengltetrazolium chloride (TZ) and dianisole bisdiphenyltetrazolium chloride (BT) ( 8 ) , but these methods do not distinguish between cortisone and hydrocortisone. There is a need for an accurate method to determine either hydrocortisone or cortisone specifically. The method reported, based on the fluorescent yellow color produced by hydrocortisone in a mixture of sulfuric and glacial acetic acids, is not specific for hydrocortisone but can be used to determine hydrocortisone in substance, in mixtures with cortisone, and in pharmaceutical preparations. REAGENTS
Unless otherwise indicated, all reagents are reagent grade. Sulfuric acid.
Glacial acetic acid. Ethyl alcohol. Mixed acid reagent. Carefully mix 4 volumes of glacial acetic acid with 6 volumes of concentrated sulfuric acid. Cool t o room temperature. Prepare standard hydrocortisone acetate solution by dissolving 50.0 mg. of hydrocortisone acetate which has been recrystallized three times from hot alcohol and dried under vacuum a t 100’ C. in 100.00 ml. of 95% alcohol. Pipet 20.0 ml. of this solution into a 100-ml. volumetric flask and dilute to the mark with alcohol. Each milliliter of this solution contains 100 y of hydrocortisone acetate. RECOMMEh DED PROCEDURE
Keigh 50.0 mg. of the sample of hydrocortisone acetate into a 100-ml. volumetric flask, dissolve in alcohol, dilute t o the mark with alcohol, and mix well. Pipet 20.0 ml. of this solution into a 100-ml. volumetric flask, and dilute t o the mark with alcohol. Measure 2.0-ml. aliquots of t h r above solution into 2.5 X 20 em. glass-stoppered test tubes. Evaporate the contents to dryness by heating in a boiling water bath. Cool the tubes t o room
Table I.
Absorption Maximum and %
1 cm. Max., 470 RIp Max., h k Mixed 1LIp Compound Mixed Acid Acid &SO4 470 175 390,445-475 Hydrocortisone alcohol Hydrocortisone acetate 470 225 390,443-475 475 45 395-400 Hydrocortisone aldehyde 0 345, 415 Cortisone alcohol No max. 0 345,415 Cortisone acetate No max. Cortisone aldehyde No max. 9 390 375-390,470 215 370, 465 Corticosterone 375, 460 A 370, 435 Desoxycorticosterone acetate 0 S o max. 3,21-Diacetoxypregnane-ll,20-dione No max. No max. 1 415, 465-475 4,5-Dihydrocortisone alcohol 6 415, 465-475 4,5-Dihydrocortisone acetate No max. 4-Bronio-17-hydroxy-2l-acetoxypregnanetrione-3,11,20 No max 1 405-420 20-Cvanoureenane-17.3.21-diol-11. . 1 405 No max. one 415 3 3.17-Dihydroxypregnane-11,20-dione 375-395 3,21-Diacetoxy-20-ketopregnanediol-
ll@,17a 21-Hydroxypregnanetrione-3,11,20 3,20-Diketo-pregnanediol-l1 m,17a Estrone Estradiol Cholesterol Ergosterol Stilhesterol Phytosterol Testosterone propionate Testosterone Calciferol 7-Dehydrocholesterol Progesterone Desoxycholic acid Lithocholic acid Cholic acid Apocholic acid
No max. 405-430 375 455-46 5 470, 510 N o max. 390,450-490 485 405,485,495 No max. 385-400 380, 510 390 No max. 385 No max. 385-395,415 385,410-415
0 23 13
415
75
450
135 6 76 265
34 4 18 29 92 3 3 2
16 13
450 440-470 365,425,450 410-415 415,490 460 410 No max. 440 380, 510 390 390 385,440 No max. 390 390,480
V O L U M E 2 7 , N O . 6, J U N E 1 9 5 5
945 produced were determined on a Cary recording spectrophotometer. These curves differed with respect to shape and position of the absorption maxima. Table I summarizes the results. Temperature and Time. The effect of temperature and time on the color formation was determined by treatment of 200 y of hydrocortisone with 10 ml. of the 3 to 2 acid mixture. Increase in temperature decreased the color formation. The color reached its maximum at room temperature after 45 minutes, and was stable for an additional 30 minutes (Table 11). Calibration Curve. A plot of the absorbancies against concentration of hydrocortisone was found to be linear and passed through the origin. Beer's law is obeyed over a suitable range of concentration, 0 to 300 y . Precision of Method. Samples of hydrocortisone acetate and hydrocortisone alcohol were assayed by the procedure described. Good reproducibility was obtained, with a standard deviation of *I%. Recovery. llixtures of hydrocortisone acetate and cortisone acetate were prepared. The amounts of mixtures taken for analysis and recovered are given in Table 111. PROCEDURE FOR HYDROCORTISONE IN OINTMEhTS
I
I
350
400
I 450
550
800 I
500
22yL Figure 1.
Absorption curves for hydrocortisone
Experimental. Substances that may he used in the preparation of ointments were tested by the procedure described above. Substances such as lanolin, liquid petrolatum, white petrolatum, white wax, beesmx, neomycin, and bacitracin were tested. Lanolin produced a reddish-brown color, the waxes produced a turbidity, and neomycin and bacitracin showed no color formation.
temperature. Pipet 10.0 ml. of the mixed acid reagent into each tube and shake vigorously for a t least 15 minutes to dissolve the residue completely in the tube. Stopper and let stand. Read the color formed in 1 hour after the addition of the mixed acid reagent in a suitable spectrophotometer or colorimeter set a t 4T0 mpwith the mixed acid as the blank. Preparation of Standards. Measure 1.0, 2.0, 3.0, and 4.0 ml. of the standard hydrocortisone solution into 2.5 X 20 cm. glassstoppered tubes and proceed in exactly the same manner as above and a t the same time as the sample is run. .o
EXPERIV E A T A L
The above procedure was developed after a study of the effect of acid concentration, temperature, and time on the reaction for hydrocortisone and 31 other similar compounds. Variations in Absorption Maximum with Increasing Acetic Acid Concentration. The color obtained from hydrocortisone in concentrated sulfuric acid shows a maximum a t 390 nip and a broad band between 445 and 475 mp (Figure 1 ) : Sulfuric acid diluted with glacial acetic acid produces a lower 390 mp niaximum and produces an absorption peak at 470 mp. The absorbance at 470 mp reaches its maximum when the sulfuric acid concentration is reduced to 60 volume % with glacial acetic acid. Additional dilution of the sulfuric acid decreases the color intensity (Figure 2). All absorptions were measured after 1 hour. Cortisone in concentrated sulfuric acid has one absorption peak a t 345 mp, one at 415 mp, and a small shoulder at 470 mp (Figure 3). Use of sulfuric acid diluted with glacial acetic acid depresses the two maxima and the shoulder at 470 mp, so that in a 60% sulfuric acid mixture the absorption at 470 mp is negligible. Figure 4 shows the absorption curves obtained when hydrocortisone and cortisone were treated i+ith a 60% sulfuric acid40% glacial acetic acid mixture (by volume) and concentrated sulfuric acid under the conditions described in the procedure. From these data the acid mixture was selected as the most favorable reagent. Hydrocortisone, cortisone, and 30 additional compounds !yere treated with 60% su1furic-40% glacial acetic acid mixture and concentrated sulfuric acid. The absorption curves of the colors
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60
1
Figure 2.
Table 11.
1 M
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7.
H,
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w
$0
so.
Effect of sulfuric acid concentration
Effect of Time on Color Formation
(200 y of hydrocortisone acetate) Time of
Standing, Minutes 15
Absorbancy, Ai7Q 0 393 0 405 0 410 0 411 0 410 0 390 0 375
30
45 60
7.5
90 120
Table 111. Recovery-of Hydrocortisone Acetate bIixture 70 Hydrocortisone Acetate
Weight Taken,
Hydrocortisone Acetate Recovered,
Y
Y
2.0
5000 5000
3.0
4.0
6.25
10.0
17.0 33.0
40.0
50.0 57.5 67.0 80.0
5000 3200
98.0
151.4 200.0
198.0
Recovery,
% 98.0 101.0 100.0 99.0
2000
201.0
100.5
600 500 400
200.0
101.0
1200 360 300 250
200.0 200.0 200.0 200.0 200.0 200.0
98.0
100.0 100.0 99,s 99.5 100.0
ANALYTICAL CHEMISTRY
946
*'"'
Investigation showed that the hydrocortisone was completely extracted from the ointment with 5070 ethyl alcohol.
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Procedure for Ointments. Weigh 500 mg. of the ointment to be tested into a 100-ml. volumetric flask. .4dd 70 ml. of 6070
1
-
**-E*
..
CORTISONE
-
He
I
HY0ROCORTISOhE-MIXED ACID
......
MIXED ACID
CORTISONE
CORTISONE
-
-
n,
SO4
HYDAOCORTISONE
-
lip SO,
SO,
CORTISONE
BOY-.
H, SO4
CORTISONE
60Y..
ne SO,
CORTISONE SOY.
H2 SO4
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350
Figure 4.
u Figure 3.
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alcohol and heat on a steam bath for 15 minutes with occasional shaking. Cool in cold water and dilute to mark with 50% alcohol. Mix thoroughly. Filter through a small dry filter, rejecting the first 10 to 20 ml. of the filtrate. Pipet an aliquot of the clear filtrate corresponding to 200 y of hydrocortisone into a large, glass-stoppered tube. Evaporate the contents of the tube to dryness by heating in a boiling water bath. Cool and proceed as described in the recommended procedure.
___-
\;
Absorption curves for cortisone
Precision and Accuracy. I n the assay of ophthalmic ointments and lotions of hydrocortisone made to contain 10 mg. of hydrocortisone per gram of ointment or lotion the standard deviation for 12 assays was 0.2 mg. per gram with an average of 10.2 nig. per gram. On an ointment made to contain 25 mg. of hydrocortisone per gram of ointment, the standard deviation for eight ass;ys was 0.4 mg. per gram with an average of 25.2 mg. per gram. DISCUS SIOY
The study of the colors obtained with the mixed acid re'tgent shows that hydrocortisone, corticosterone, Zl-hydro\ypregnanetrione-3,l 1,2O-estradiol, estrone, and stilbestrol produce a fluorescent yellow color. These findings suggest that the fluorescent reaction can be of analytical value in identifying these compounds. A study of the difference in the fluorescence spectra of hydrocortisone and corticosterone could be used in analyses of these closely related substances. It has been reported ( 3 ) that the differences in fluorescence spectra of estrone and estradiol are sufficient to permit the optical measurement of one compound in the presence of the other. The absorption spectra of steroids treated with sulfui ic acid differ from the spectra of the same steroids treated with sulfuricacetic acid reagents. KnoLsledge of the spectral differences of
I
400
__-_ *. .", '
500
450
550
600
m
Comparison of hydrocortisone and cortisone curves
steroids in various reagents may prove valuable as a basis for the development of future quantitative steroid techniques. The procedures given for hydrocortisone are based on the requirement of about 200 y of hydrocortisone for development of sufficient intensity of color to minimize instrumental error. By use of cell paths greater than 1 em. in depth this sensitivity c u i be increased. The outlined procedure has several advantages: The tept distinguishes between hydrocortisone and cortisone, hydrocortisone can be determined in the presence of cortisone, common laboratory reagents are used, sulfuric acid and glacial acetic acid, and a stable colorless blank is obtained. REFERENCES
(1) Banes, D., J . A m . Pharin. dssoc.. Sci. Ed., 42, 669 (1953). (2) Clark, I., AIerck Institute of Therapeutic Research, private communication. (3) Dhere, C., and Lasrt, L., Compt. read., 224, 681 (1947). SOC.Ezptl. Biol. M e d . , 68, 181 (1949). (4) Finklestein, RI., PTOC. ( 5 ) Goldzieher, J. W., EndocrimZog~/,53, 527 (1953). (e) Goldzieher, J. W., J . Lab. Cliii. Afed.. 33, 251 (1948). (7) Goldzieher, J. W., Bodenchuk, J. 31., and Nolan, P., ASAL. CHEM.,26, 853 (1954). ( 8 ) AIader, W. J., and Buck, R. R., Ibid., 24, 666 (1952). (9) AIeischer, K., Helv. Chim. A c t a , 29, 743 (1946). (10) Pesos, hl., Bull. soe. chim., France, 16, 5Oi (1949). (11) Porter, C. C., and Silber, R. H., J . Bid. Chem., 185, 201 (1950). (12) Reichstein, T., and Shoppel, C. W., Vitamins and Hormones, 1, 345 (1943). (13) Sweat, A I . L., ANAL.CHEY.,26, 773 (1954). (14) Tauber, H., Ibid., 24, 1494 (1952). (15) Umberger, E. J., and Curtis, J. 31., 6.Biol. Chem., 178, 275 (1949). (16) Winterst,einer, O., and Pfeffner, J. J., Ibid., 116, 291 (1936). (17) Zafforoni, A.. J . A m . Chem. Soc.. 72, 3828 (1950). RECEITEDfor reviex November 18,1954, .iccepted January 29, 1955. Presented before the North Jersey ASIERIC.IS CrrEsfrcaL SOCIETY Aleetinn-in;\Iiniature, January 24, 1955.
