Spectrophotometric Determination of A4,3-Ketosteroids DENIS ABELSON' and PHILIP K. BONDY Departments o f Medicine and Physiology, Yale University School o f Medicine, New Haven, Conn.

1 method is described for the spectrophotometric de- termination of A4-3-ketosteroids. These substances react with potassium tert-butoxide in tert-butyl alcohol to produce a yellow color with absorption maxima at 260 and 385 mp. The absorbance at 385 mp varies with steroid concentration in accordance with Beer's law. The method is sensitive to 1 to 2y of steroid. Estriol, estradiol, and estrone show- absorption maxima at 250 and 310 mp with the potassium butoxide reagent.

not usually exceed that of the reagent blank; in the few cases where a small reading was obtained, the absorption maximum was a t a shorter wave length than 385 mp. In the case of choles- tane-3,6-dione, however, an absorption maximum was found a t 385 mp, the molar extinction coefficient being of the same order as that obtained with A4,3-ketones.

7

OST quantitative cheniical procedures for the determina- M tion of corticosteroids depend on properties of either the side chain or the A4,3-ketone grouping. In addition, numerous qualitative or semiquantitative spot tests exist for the detection of corticosteroids of paper chromatograms. It has recently been shon-n (1) that the sodium hydroxide spot test for A4,3-keto- steroids ( 2 ) may be adapted to the quantitative fluorometric esti- mation of these compounds in anhydrous solution. The spot test is based on the production of an orange-yellow fluorescence in ultraviolet light when A4,3-ketosteroids on paper chromato- grams are sprayed vi th 10% sodium hydroxide solution and allorred to dry. For the quantitative reaction the most suitable alkali was found to be potassium tert-butoxide dissolved in tert- butyl alcohol. A yellow color nas observed in the solution when concentrations greater than about 10 y of A4,3-ketosteroid per ml. of reagent were used. This finding has been utilized in a spectrophotometric procedure for the quantitative anal) sis of A4,3-ketosteroids.

EXPERIMENTAL

Reagent. Approximately 1 gram of metallic potassium is heated with tert-butyl alcohol for about 2 minutes, then washed with several rinses of tert-butyl alcohol. It is then refluxed in all- glass apparatus with about 40 ml. of tert-butyl alcohol under nitrogen until completely dissolved. The reagent is stored in a vacuum desiccator until use.

Preparation of Steroid Samples. The crystalline steroids were analyzed as received without further purification. When J T , ~ ~ , measurements were to be made, the steroids were dried for 2 11 eeks over calcium chloride in a vacuum desiccator, weighed out in approximately 10-mg. quantities on an analytical balance, and made up to volume in volumetric flasks with absolute ethyl alco- hol. The E,,,. of the alcoholic solutions a t 240 to 242 mp was compared with published values (6). In every case the observed E,,,. agreed with the published figures within 5%.

Apparatus. Spectrophotometric measurements were made with a Beckman DU spectrophotometer, using 1.000-cm. matched silica cells. Constant sensitivity was maintained by varying the d i t width.

RESIJLTS

The absorption spectra of steroids containing the A4,3-ketone configuration shoxed maxima a t 260 and 385 mp (Figure 1). Several steroids lacking the A4,3-ketosteroid configuration ab- sorbed in the region from 240 to 260 mp, but, with the exception of cholestane-3,6-dione, only those steroids which contained the A4,3-ketone grouping showed a maximiini a t 385 mp. The maxi- mum a t 385 mp was unchanged in position after standing for 2 hours a t room temperature, whereas the maximum a t the lon-er wave lengths shifted in some cases as much as 10 mp in either direction on standing. Table I gives the absorbances of a num- her of steroids at 385 mp, using a concentration of 20 y per ml. In the absence of the A4,3-ketone grouping, the absorbance did

1 Present address, Department of Medicine, Postgraduate RIedlcal School, Hammersmith Hospital, London England.

240 280 320 360 400 440

W A V E L E N G T H , rn)

Figure 1. iibsorption spectra of l'i-hydroxycorti- costerone (22.5 y per ml.) in 0.3Npotassium tert-butox-

ide reagent Lower curve made after 10 minutes and upper curve made after

2 hours at room temperature

The rate of color development behaved in the same general manner as the fluorescence; thus 17-hydrosycorticosterone waf the slowest steroid to reach both maximum fluorescence and maximum absorption, whereas 11-dehydrocorticosterone was the most rapid. There was more variation in the rate of color development than fluorescence development, and the latter in most cases R-as more rapid.

Table I . Absorbance at 385 mp Produced after 1 Hour by 10 y of Steroids in 0.5 M1. of 0.1N Potassium tert-Butoxide

Solution Absorbancea

Saturated Androstane-3 17-dione 0 024 (inax. = 335 mr )

Pregnane-3a,ZOa-diol Pregnane-17a,21-d1ol,3,11,20-trione (pregnanediol) (dihydro- 0 0 006 062 (mar. = 342 mr ) cortisone)

hydrocortisone) 0 .006 Pregnane-3u,l7a,21-triol,ll,2O-~Iione (tetra-

C holestane-3,B-dione 0.311 (max. = 386 m r ) As-enes

Cholesterol 0 000

sterone) 0.000 As-Androstene-3@-ol, 17-one (dehydroepiandro-

Al-ene-3-ones

Ah-ene-3-ones

Aj-ene-7-ones

A8 (Q>-ene-:-ones

A9 11 Lene- 1 2-ones

AI-Cholestene-%one 0.000

Ah-Androstene-3, li-dione 0.460

7-Ketocholesteryl acetate 0 014

A S 191, 92-Ergostadiene-3-ol-i-one 0.023 (mar. = 265 m r )

3-Hydroxy-12-keto-AQ~~~~-cholenic acid 0 000

Read against butoxide blank in silica microcuvettes with 1-em. light path.

1922

V O L U M E 2 8 , NO. 1 2 , D E C E M B E R 1 9 5 6 1923

The rate of color development was also found to depend on base concentration in the same way as the rate of fluorescence development. In strongly alkaline solutions color development was more rapid, whereas in weaker solutions it was retarded (Figure 2). For routine purposes absorbance was most con- veniently measured a t 1 hour, using 0.4N butoxide solution.

For a given steroid, if the base concentration and time of color development were kept constant, the absorbance a t 385 mp varied with steroid concentration in accordance with Beer's law. Values of E,,,. for a number of A4,3-ketosteroids'in 0.4.V butoxide are given in Table 11. Because of the variation in both rate of color development and E,,,. values, the method is not suitable for analyzing mixtures of steroids. With single steroids the technique is highly reproducible, as the standard

l i ! Lu c) z 4 m a 0 v)

4 m

, ;\,-, , , , , I 200 240 280 320 360

W A V E L E N G T H , m g

Figure 3. Absorption spectrum of estradiol (26.3 y per ml.) in 0.3N potassium tert-butoxide reagent after 2

hours at room temperature

deviation calculated from six replicates of a single steroid solu- tion was 3 ~ 2 . 5 7 ~ of the mean value.

Estradiol, estrone, and estriol, R-hich normally absorb maxi- mally a t 280 mp in neutral ethyl alcohol, showed absorption peaks

a t 250 mp and 305 to 310 mp in the potassium tert-butoxide S O ~ U -

tion (Figure 3). The E,,,, a t 310 mp after 2 hours in 0.3LY potassium butoxide was approximately 3580 for estradiol, 3800 for estrone, and 3060 for estriol. At 250 mp under identical con- ditions, the E,,,. for estradiol was 8820, for estrone 9360, and for estriol 7860.

DISCUSSIO\

The color produced by 14,3-ketosteroids in potassium tert- butoxide appears to have the same significance as the fluorescence that develops in a parallel fashion, and it is possible that both are properties of the same structural grouping. The nature of this structural grouping has not yet been elucidated.

Results similar to those reported above n-ere also ohtained by Cross, Eisen, and Kedersha ( J ) , n-110 noted a maximum near 375 mp for a number of li,3-ketosteroids after heating in 1.1-1- aqueous tetramethylammonium hydroxide. hIeL-er found a maximum near 380 mp with 14,3-ketones on exposure to alkaline ethyl alcohol a t 23' andlor 60" C. ( 7 ) . In both studies the maxima were much less intense than those reported here, and a t a slightly different wave length. These differences are prob- ably related to the different solvents used, and particularly to the use of aqueous solutions. Both the fluorescence and the color reaction with the butoxide reagent are strongly inhibited in the presence of water; indeed. the presence of as little as 0.1%

Table 11. Values of Emax.* for Some A4-3-Ketosteroids in O.PN Potassium tert-Butoxide Solution

Testosterone 7,200 Progesterone 7,200 1 1-Desoxycorticosterone 5 600 11-Dehydrocorticosterone 7 800 17-Hydroxy, 11-dehydrocorticosterone 8 ,760 17-Hydroxy,ll-dehydrocorticosterone acetate 8 ,100 Corticosterone 11,000 17-Hydroxycorticosterone 10,300

Emax.

a Development of color was followed in a Coleman Junior spectrophotom- eter until absorbance had reached a maximum. The solution was then trans- ferred t o a 1.0-cm. silica microcuvette and the absorbance measured a t 385 mp in a Beckman D U spectraphotomiter. Emaa. = absorbance a t 385 me in 0.4N potassium butoxide solution/mole /liter/l-cm. light path.

1924 A N A L Y T I C A L C H E M I S T R Y

for Biological Research, and John J. Schneider, Jefferson Medical School.

LITERATURE CITED

(1) Abelson, D., Bondy, P. K., Arch. Bwchem. and Biophys. , 57, 208

(2) Bush, I. E., Biochem. J . 50, 370 (1952). (3) Callow, R. K., Ibid., 30, 907 (1936). (4) Cross, J. RI., Eisen, H. E , Kedersha, R. G., -4x.4~. CHEX. 24,

(5) Dorfman, L., Chem. Reus. 53, 47 (1953). (6) Hamburger, C., Acta Physiol. Latinoamer 3, 109 (1953). (7) hleyer, A., J . Org. Chem. 20, 1240 (1955). (8) Pedersen-Bjergaard, K., Schou, -4 S., Quart. J . Phnrmacol. 8 , 669

RECEIVED for review December 17, 1955. Accepted June 22, 1956. Kork supported by Research Grant -4 254(C3) from the National Institutes of Arthritis and Metabolic Diseases, National Institutes of Health, U. S. Public Health Service.

(1955).

1049 (1952).

(1935).

water in the system produces appreciable changes in the relative intensity and location of the ultraviolet peaks.

The presence of a peak near 310 mp in alkaline solutions of estrogens has previously been reported by Pedersen-Bjergaard and Schou ( 8 ) , Hamburger ( 6 ) , and Callow (S), and attributed by the last to the formation of a phenoxy salt in ring A. The peak a t 250 mp has not previously been reported, to the authors' knowledge, although Hamburger's curves show a continuous increase in absorbance as the wave length decreased to 250 mw,

ACKNOWLEDGMENT

The steroids used in the study were kindly supplied by Merck & Co., Inc., Upjohn Co., G. D. Searle and Co., Schering Corp., Sloan-Kettering Institute, Andre Sfeyer, Worcester Foundation

Hydrocarbons in the 126" to 132" C. Fraction of Petroleum M O R T O N B. EPSTEIN', C H A R L E S B. WILLINGHAM2, BEVERIDGE 1. M A I R , and FREDERICK D. ROSSlNl Carnegie lnstitute of Technology, Pittsburgh 13, Pa.

Seven hydrocarbons were found in the fraction of petroleum normally boiling between 126" and 232" C., which constitutes approximately 0.68% by volume of the representative petroleum that has been under in- vestigation for many pears by the API Research Project 6. These compounds were concentrated by extended use of the fractionating processes of distillation (both regular and azeotropic) and adsorption. The corn- pounds were identified by infrared and mass spectro- metric examination, coupled with measurements of the simple physical properties. The names and estimated relative amounts of the seven compounds in this frac- tion of petroleum, given in decreasing order of quantity, are: ethylcyclohexane, 53; 1-trans-2-cis-3-trans-4- tetramethylcyclopentane, 16; 1-cis-2-dimethylcyclo- hexane, 9; n-propylcyclopentane, 9; l-methyl-cis-2- ethylcyclopentane, 6; 2,3,Strimeth>-lhexane, 5 ; and isopropylcyclopentane, 2.

S PART of the continuing nork of the American Petroleum A Institute Research Project 6 on the composition of its rep- resentative petroleum ( 7 ) , analysis has been completed of the h> - drocarbons in that fraction of the petroleum normally boiling between 126" and 132' C., which constitutes approximatel\ 0.68% by volume of the original crude. The status of this ma- terial prior to the present investigation is described in previouq papers ( 4 , 6 ) . The details of the analysis of the hydrocarbons in the adjacent lower-boiling fraction of this petroleum are given in another paper (3).

DETAILS OF PROCESSIhG

All of the material from the previous nork, except a small amount of ethylcyclohexane, was blended and distilled to obtain in one lot all of the material from the original petroleum remaining in the range boiling normally from 126" to 132' C. The results of this distillation are given in Figure 1, which shows the location with respect t o boiling point and refractive index of the com- pounds found in this material. It is apparent that the first portion of the distillate consisted largely of n-octane, which nor-

1 Present address, Onyx Oil and Chemical Co , Jersey City, N. J. * Present address, Mellon Institute of Industrial Research, Pittsburgh 13,

Pa.

mally boils slightly below 126" C., and the portion just before the tail end consisted largely of ethylcyclohexane, which normally boils just below 132" C. The present analysis includes ethyl- cyclohexane but not n-octane, which is covered in the analysis of the next lower-boiling material (3).

The material was processed by distillation (regular, azeotrope, and at reduced pressure) and by adsorption in a sequence de- signed to concentrate the individual hydrocarbon compounds so that they could be identified m-ith reasonable certainty. The amounts of the individual compounds xere computed from the results of spectrographic analyses made on appropriate inter- mediate and final fractions by the Research Laboratory of the Humbleboil and Refining Co.

The processing of the material by distillation, following the distillation shown in Figure 1, is summarized as follows (see 8 for details of the distilling apparatus and procedures):

Lot 1-B, from Figure 1, Part B, 1870 ml., was distilled a t 725 mm. of mercury.

Lot 2-I-C, from Figure 2, Part I-C, 1460 mi., was distilled a t 56 mm. of mercury. The results are shown in Figure 2, Part 11.

Lot 2-11-A, from Figure 2, Part II-.4, 505 ml., was distilled azeotropically with ethylene glycol monoethyl ether (Cellosolve) a t i25 mm. of mercury. The results are shonn in Figure 2, Part 111.

Lot 2-11-B, from Figure 2, Part 11-B, 400 ml., was also dis- tilled azeotropically with ethylene glycol monoethyl ether at 725 mm. of mercury. The results are shown in Figure 2, Part IV.

Lot 2-11-C plus 2-IV-A, from Figure 2, Part 11-C plus Part IV-A, 580 ml., was also distilled azeotropically with ethylene glycol monoethyl ether a t 725 mm. of mercury. The results are shown in Figure 2, Part V.

Lot 2-111-B, from Figure 2, Part 111-B, 285 ml., was distilled at 725 mm. of mercury. The results are shown in Figure 3, Part I.

Lots 3-I-A and 3-I-C, from Figure 3, Parts I-A and I-C, plus cj-cloparaffin concentrates separated from Lot 1-A, from Figure 1, and Lot 2-111-A, from Figure 2, total charge of 250 ml., were dis- tilled regularly a t 725 mm. of mercury. The results are shown in Figure 3, Part 11.

Lots 3-I-B and 3-11-B, from Figure 3, Parts I-B and 11-B, 205 ml., were distilled axeotropically a t 725 mm. of mercury. The results are shown in Figure 3, Part 111.

Lot 2-V-A, from Figure 2, was further fractionated by adsorp- tion with silica gel in a tall column (1 by 790 cm.) to give the re- sults shown in Figure 4 (see 6 for details regarding the apparatus and procedure for fractionation by adsorption).

In the foregoing, the lots of material are identified with ref- erence to the figure in which the results are plotted. Thus, Lot 2-I-B refers to Figure 2, Part I, Portion B.

The results are s h o m in Figure 2, Part I.