ANALYTICAL CHEMISTRY
666 Table I. Sample NO.
1
2
3 4 5
6 7 8
photometric analysis of synthetic eugenol-isoeugenol samples are presented in Tablc I.
Comparison of Known and Calculated Concentrations for Eugenol and Isoeugenol in Synthetic Samples
Eugenol, 3Iole,’Liter Known Calcd Error. 7” -0 8 306 X 10-4 3.02 X lo-‘ -0 3 2.41x 10-4 2 . 4 0 x I O - & -0 3 2 14 X 10-4 2.13 X IO-‘ -1.8 1.73 X 10-4 1.70 X 10-6 1.51 X 1.42 X -6 1 9.14 X 10-8 9.37 X 10-6 +2 0 2.55 X 10-a 2.56 X 10-5 +O j 2 26 X 10-5 2 . 0 3 X 10-5 -9 5
LITERATURE CITED
Isoeugenol, hlole/Liter Known Calcd. Error, % 7.44 X 10-6 8 4 5 X 10-6 4-7 8 1 . 2 6 x io-j 1 . 3 1 x 10-5 +3 i,i5 x 10-5 i 20 x 10-5 +5.; 1.83 X 10-5 1 . 8 0 X 10-5 -1 I 2.59 X 10-5 2 . 5 9 X 10-5 00 4.86 X 10-5 4 ( 9 X 10-5 -1 3
5.45 X 10-6 5.37 x 10-5 3.84 X 10-5 3 87 X 10-6
(1) Doub, L., and Vandenbelt J If, J . .4m C h e m Soc., 69, 2714 (1947). (2) Ibzd., 71, 2414 (1949).
(3) Guenther, E., “The Essential Oils Vol I, p 291 New York, D. Van Kostrand Co , 1949 (4) Guenther, E , and Langenau, E I: A s i ~CHEV 21, 202 (1949). ( 5 ) Ibzd., 22, 210 (1950). (6) Ibid., 23, 217 (1951). (7) llellon, lf, G., “;inalytical Absorption Spectroscopy,” p. 191. S e w York, John %ley Br Sons.
-l.R
t0.8
1950. cI
=
a;5+a42sa ap54a:s2
The analytical results
Of
- a~s2a~2s4 - a:s4a3s2
(5)
the simukaneous ultraviolet sPectr0-
(8) Ihid., p. 370. (9) Uyeo, S.,Miwsa, T., and Sakaniahi, RI., J . Chem. Soc. J a p a n . 64, 659 (1943). RECEIVED f o r review
J U I ~23,
1051. Accepted January 24, 1952.
Colorimetric Determination of Cortisone and Related Keto1 Steroids W. J . 3I-iDER AND R . R . RUCK Chemical Control Division, Lklerck& Co., I n c . , Rahway, .\-.J . U ith the advent of the commercial production of cortisone rapid, accurate, and sensitive methods were needed for the quantitative and qualitative determination of this product in order to control plant production, dosage in pharmaceutical preparations, and body levels of this important ketol steroid. A colorimetric method based on the reducing properties of the ketol moiety in tetrazolium salts was investigated. A method is presented by which as little as 10 micrograms of cortisone or related ketol steroids can be determined and by obvious modifications much smaller quantities detected. This analytical procedure has been of help in production, control, and research on steroids.
0
Tu’ THE basis of the current knowledge of steroids, anti-
rheumatic activity is dependent upon specific chemical structures. The presenceof a ketone a t carbon 20 adjacent tc? the primary hydroxyl group a t carbon 21 and an unsaturated ketone group a t pdsitions 3, 4, and 5 in adrenal steroids is essential to cortical activity ( 3 ) . Antirheumatic activity requires in addition a hydroxyl group or a ketone group at, carbon 11 and a hydroxyl group a t carbon 17. Cortisone acet)at>e(11-dehydro-17-hydroxycortjicosterone-21-acetate) and 17-hydroxycorticosterone are the onh. known steroids that meet these requirements. The ketol group imparts to the steroid molecule reducing properties similar to those of fructose, which contains the same aketol group. Tetrazolium salts yield, upon reduction, deep red water-insoluble pigments knou-n as formazans ( I ) . The tetrazolium chlorides have a reduction poteiitinl of about -0.08 volt (2). Rutenburg et al. (4)have shom-n that a stable blue tiiforniazan is obtained from the reduction of 3,3’-dianisole-bis-4,4’-(3,5dipheny1)tetrazolium chloride. .Ilcoholic solutions of steroids which contain the primary a-keto1 group reduce tetsazolium salts in the presence of tetramethyl ammonium hydroside and form colored solutions. I n the case of 2,3,5-triphenyltetrazoliuni chloride and 3,3-dianisole-bis-4,4’-(3,~-diphenyl)tetrazoliiim chloride the color produced follows Lambert-Beer’s law over a suitable concentration range, as shown in Figure 1. The absorption curves for the formazan obtained from 2,3,5-triphenyltetrazolium chloride (TZ) and the diformazan from the dianisole bisdiphenyltetrazolium chloride (BT) and cortisone aceCate are given in Figure 2. As expected, the molecular absorption of the
diformazan from cortisone acetate and dianisole bisdiphenyltetrazolium chloride is twice that of the forniazan of cortisone acetat’e and 2,3,5-triphenyltetrazolium chloride. The laboratory procedure as reported can be used to determine cortisone acetate in a concentration range of 0.01 to 0.17 mg. per ml. By obvious modifications of the method, much snialler example, the dianisole bisdiamounts could he detectcd-for
0.9 0.8
‘ / . ’
0.7 .
BT
M G. c b RTI s ON E A C E T A T E
Figure 1. Absorbancy
V O L U M E 2 4 , NO. 4, A P R I L 1 9 5 2
667
phenyltetrazolium chloride reagent has been found to be excellent lor the quantitative determination of ketol steroids in paper ~~hroiiiatograpliy.A separate paper will cover this phase of the 73 or]\.
-4 total of l i steroids and related coiiipouritls were ;tudied. Five of these compouiids (Table I ) coutained t.he a-keto1 group :md developed color lr-ith both dianisole bisdiphen?;ltetrazoliuni 1,hloride and 2,8,5-triphenyltetr~izoliumchloride. The reinaining conipounds did not reduce the renyrnta. One could predict that i n addition to the five that produced color, 0tht.r ketol etei,oitls, .such as 17-hydrosycorticosterone (Compound F), dihydrocorticosteronc, 6-tlehydrocortisoiie, arid 21-acetosypregnenolone, would behave like mrtisone: however, these steroids have not iwen investigateti.
Color w i t h Yiibstitution -7% R T Compound Carbon 20 Carbon 21 Cortisone acetate c= 0 11-Desoxycorticosterone acetate c= 0 ---CH~OOCCHI Corticosterone c= 0 -CHzOH 17-Hydroxy-1 l-desoxycorticoc= 0 -CHzOOCCHa sterone acetate 11-Dehydrocorticosterone ace~ j t-a3t-eH y d r o x y p r e g n e n e - ~ O - o n e C= 0 -CHzOOCC Hr e= 0 HI A‘-Preenene-17.20.2 l-trlol-3rrTofI one A4-Androstene-3,11,17-trione l‘-Androstene-3,17-dione Estradiol Estrone c= 0 Progestrone ---CHI Methyltestosterone Testosterone propionate Ethinyl testosterone Cholic acid Desoxychloric acid
-
-
+ + +
--c
-
+
The 2,3,5-triphenyl tetrazolium c,lrloride solut’ion is prepared in a similar fashion. Standard cortisone solution is prepared by dissolving 25 ing. of cortisone acetate which has been recrystallized three times from hot alcohol and dried under vacuum a t 100” C. in 100 inl. of 95% alcohol; 50 ml. of this solution are diluted to 100 nil. Preparation of Sample. For assaying the pure chemical, a solution containing 120 to 130 micrograms of cortisone acetate in 10 ml. of ethyl alcohol is prepared. For tablets containing cortisone acetate, the tablets are ground and an aliquot is used according to standard procedures. The powder is estracted with absolute ethyl alcohol or isopropyl alcohol to separate t8hesoluble cortisone acetate from interfering substances such as lactose. To prepare ointments for analysis, the ointment is suspended in hot absolute alcohol, and the alcohol is separated by decantation and diluted to volume.
0.8
0.7
5 0.6 z
3 0.5 2 0.4 K
0
0.3
0.2
0
Rate of Color Formation
-
INTERFEREYCE
0.1
Table I.
440
480
Figure 2.
520
560
600 0.8 L
.4bsorption Curves 0.7
-
,* - .:
--- .--=- 7-r-7.r-F--: -7r.z :
7.7-7 : -7
Iieducing sugars \vi11 also produce a color with tetrazoliuin chlorides and must be separated from samples containing ketol steroids. Because cortisone acetate is soluble in absolute ethyl alcohol or isopropyl alcohol and most reducing sugars are not, the cortisone acetate may be separated from such substances (such as lactose) by alcohol extraction prior to colorimetric estimation. RATES O F COLOR FORMATION
The rate of color formation and the aniount of color produced by the ketol steroids investigated varied, as illustrated in Figure 3. Cortisone a,cetate reached its maximuni color in 15 niiiiutee ant1 color was stable for a t least 1 hour. All the ketol steroids reached their maximum color in 35 minutes. The two 17-hydroxysteroids produced less color than the three keto1 steroids without the 17-hyd:oxyl group, and conversely, the two 11-desosysteroids produced less color than t,he two 11h ydroxysteroids. PRECISION AND ACCURACY
In the assay of an ophthalmic ointment of cortisone acetate n i a t l ~to contain 25 mg. of cortisone acetate per gram of ointment the standard deviation for eight assays was 0.3 nig. per gram airh an average of 25.3 mg. per gram. The standard deviation for cortisone acetate tablets was 0.3 mg. for ten determinations with an average of 27.5 mg. per tablet. PROCEDURE
Reagents. Dilute tetramethyl ammonium hydroxide is prepared by diluting 10 ml. of the 10% aqueous solution to 100 ml. with 95% alcohol. Dianisole-bis-4,4’-(3,5-diphenyl)tetrazoliumchloride solution is prepared by dissolving 500 mg. in 100 nil. of 95% alcohol. This solution is protected from light and prepared fresh daily.
TIME I N MINUTES
Figure 3.
Rate of Color Formation
Color Development. An aliquot of the sample containing from 120 to 130 micrograms of cortisone acetate in 10 nil. of alcohol is added to one of three glass-stoppered test tubes. To the second tube, 125 micrograms of standard are added and all three are made up to 10 ml. with ethyl alcohol. To each is added 1 nil. of the diluted tetramethyl ammonium hydroxide solution followed by 1 ml. of the dianisole bisdiphenyltetraaolium chloride solution. After the sample and blank have stood for 15 to 20 minutee, a suitable photoelectric colorimeter is nulled using the blank tube. The absorbancy is measured on the sample and the standard a t 510 inp, and the concentration is calculated in the usual manner. LITERATURE CITED
(1) Kuhn, R., and Jerchel, D., Ber., 74, 941 (1941). (2) Zbid., p. 949. (3) Pincus, G., and Thimann, K. V., “The Hormones,” 5’01, 1, p. 333, (4)
Kew York, Academic Press, 1948. Rutenburg, A. M., Gofstein, R., and Seligman, -k. SI.,Cancer Research, 10, 113 (1950).
review October 4 , 1951. Accepted January 2 6 , 19.52. Presented a t the Meeting-in-Miniature of the S o i t h Jersey Section, AMERICAN C H E 3 I I C A L SOCIETY, January 8, 19.51. R E C E I V E Dfor
