dehted to Eva A . Smitli for assistance in the experimental work and to Marelynn AT. T e i s s for checking the calculations. LITERATURE CITED
(I) Bergmann, E. D., Crane, F. E., FUOSS, R . AI., J . Am. Chenz. Soc.
74,5981 (1952). 12) Blair, A. J., Pantonv, D. A., A n a l . Chim. Acia 13, 3 (-1955). (3) Cooper, S. S.,AXAL. CHE1f. 29, 446 (1957). (4) Crane, F. E., Ibid., 28, 1794 (1956). (5) Crane, F. E., A n a l . China. S c t a 16, 370 (1957). ( 6 ) Crane, F. E., Fuoss, R. AI., AXAL. CHEM.26, 1651 (1954). (7) Dal Nogare, S., Ibid., 28, 903 (1956). '
(8) Engelbrecht, R. X.,LIcCoy, F. 8 . ,
Ibid.,28, 1772 (1956).
(9) Feigl, F., "Chemistry of Specific, Selective and Sensitive Reactions," wv. 8-10. Academic Press. ?;eT York, 1949. (10) Feigl, F., Caldas, -4.,Mzkrochim. Acta 44, 1310 (1956). (11) Feigl, F., Feigl, H. E., Ibzd., 39, 86 (1954). (12) Foucrv, T., J . pharnz. chim. 20. 116 (1934). ' (13) Geilmann, IT.,Gebauhr, IT.,Z. anal. Chenz. 142, 247 (1954). (14) Close, G. H., Olson, B., ChemistAnalyst 43, 81 (1954). (15) Koszegi, D., Salgo, E., 2. anal. Chenz. 130, 403 (1950). (16) Krumholz, P., Watzek, H., dlikrochenzie 19, 55 (1935). ~
(17) Lee, C. C., Spinks, J. IT. T., Can. J . Chcm. 32, 1005 (1954). (18) Likhosherstov, M. V., J . Gen. Chem. (C.S.S.R.) 3, 164 (1933). 119) Schall. E. D.. ASAL.Cmar. 29. 1046 (1957). (20) Stein, H. K., Hoppe-Seyler's 2. physiol. Chem. 288, 125 (1951). (21) Steiner, M., Kamienski, E. S., Satur~zssenschaSten40,483 (1953). 122) Kendlandt. W. ITT.TT.. A n a l . Chzm. Acta 16, 218 (1957). RECEIVED for review August 27, 1957. Accepted A4pril 23, 1958. Presented in part before the Analytical Division, Meeting-in-Miniature, North Jersey Section, ACS, South Orange, N. J., January 27, 1958.
Determination of Steroid Alcohols with Acetic Anhydride-C-14 VINCENT P. HOLLANDER and JULIA VINECOUR University of Virginia, School of Medicine, Charloftesville, Vu.
b A simple procedure for the measurement of steroid alcohols by acetylation with acetic anhydride-C-14 i s described. Application to pure compounds or partially purified mixtures i s intended. Preparation of the radioactive acetates permits separation, identification, and quantitation in submicrogram amounts. Excess acetic anhydride can be recovered, to decrease the cost of the analysis. Androsterone, dehydroepiandrosterone, cortisone, and corticosterone can be measured singly or in a mixture. Hydrocortisone and corticosterone can be recovered from plasma. Androsterone and dehydroepiandrosterone cannot b e adequately recovered from plasma b y this procedure. The procedure is capable of submicrogram determination of steroid alcohols in favorable cases, where radiopurity of the derivative can be obtained.
T
laboratory is engaged in the separation and estimation of submicrogram quantities of steroids in crude chromatographic fractions of blood and tissue. The radioactive derivative niethod of Keston and colleagues (8) lends itself to this problem because of high sensitivity and manipulative ease. I n general, the method involves the coupling of the unknown u ith a reagent of high specific activity. After destruction of excess reagent and addition of nonradioactive product, the derivative is purified. Determination of the HIS
specific activity of the derivative readily permits calculation of the amount in the original sample. Bojesen, Keston, and Carsiotis (3, 4) use the iodine-131 anhydride of p iodobenzenesulfonic acid as a reagent for the determination of corticoids. Avivi and colleagurs ( 1 ) and Berliner ( 2 ) used radioactive acetic anhydride for determination of steroid alcohols. This paper describes the preparation of acetic anhydride-C-14 of high specific activity and its use as an analytical reagent. MATERIALS A N D METHODS
Sodium acetate-1-C-14 was prepared in the usual manner from barium carbonate-C-14. S o carrier barium rarbonate n a s added. in order to achieve a yxcific activity of 8 to 10 me. per mniole. The product n-as rendered anhydrous by solution of the >odium acetate in dry methanol, evaporation of the solvent with dry nitrogen, and continuous evacuation of the container a t mni. of mercury for 12 hourq. During this process, the tube containing the product was maintained a t TO" C. by means of an air bath. Acetic Anhydride-C-14. Acetic anhydride-C-14 of 1 me. per mniole specific activity or lower lvas prepared from sodium acetate and phthallyl chloride A mixture of 164 mg. of sodium acetate-C-14 and 0.15 ml. of redistilled phthallyl chloride was sealed in a 10 x 75 mm. borosilicate test tube and heated for 2 hours in an electric oven a t 70" C. The tube was cooled to room temperature and opened n-ith care in a hood. and 0.2 ml. of dry
benzene n-as added by means of a syringe and fine needle. The tube was placed in a suitable ground-joint vessel for attachment to the high vacuum line and distilled a t room temperature into a small U-trap containing 10 mg. of anhydrous nonradioactive sodium acetate. The sodium acetate removes traces of acetyl chloride. The product was then distilled into a second t r a p and diluted by the addition of nonradioactive acetic anhydride and sufficient d r y benzene t o make a 2 to 47, qolution of acetic anhydride. This benzene solution n-as stable. The yield was 90%. Nonradioactive acetic anhydride prepared by this reaction contained a trace of unidentified impurity which boiled a t 130' C. S o residue was observed after room t m p e r a t u r e evaporation of 1 ml. of this material, which gave a negative or very faint test for halogen. This product was entirely satisfactory for determination and separation of microgram quantities of steroid. However, n-ith 0.05, samples the acetates could not be freed of a persistent radioactive impurity nhich interfered with the analysis. For submicrogram analysis, acetic anhydride-C-14 prepared by exchange ( 1 ) was satisfactory. This wac: prepared by heating a mixture of 164 nig. of sodium acetate-lC-14 (8 to 10 me. per mniole) and 0.10 nil. of nonradioactive acetic anhydride in a sealed tube a t 100" C. for 12 hours. The product was then distilled on the high vacuum line and diluted nit11 dry benzene t o a concentration of VOL. 30, NO. 8, AUGUST 1958
1429
2 to 5%. The exact concentration was not determined. Benzene was distilled over sodium. Pyridine was distilled over barium oxide. Perbenzoic acid was prepared by Hickinbottom’s method (6). Radioactivity was determined in a windowless flow gas counter on samples of less than 100 y by plating on stainless steel planchets. Samples were counted to 2% statistical accuracy.
Table
sample No. 1
2 3 4 5
6
7 8
Procedure for Determination of 0.2 to 3 y of Cortisone. The cortisone
was measured by pipetting a standard alcoholic solution into a small glass test tube and evaporating the solvent under a stream of air. The samples were dried in a vacuum desiccator overnight. To each sample were added 10 pl. of dry pyridine and 40 pl. of a 2% solution of acetic anhydride-C-14 in benzene. Tubes were stoppered and allowed to stand overnight a t room temperature. On the following day, the stoppers were removed and the tubes placed in a glass desiccator 4 inches in diameter, containing 10 to 15 ml. of concentrated sulfuric acid in a small beaker. The excess reagents were allowed to distill a t room temperature into the sulfuric acid for 24 to 48 hours. Each tube was subsequently considered contaminated on the outside and handled with forceps. Carrier cortisone acetate was added as an alcoholic solution containing approximately 200 y of cortisone acetate. The reaction mixture was dissolved in a small volume of ethyl acetate and washed twice each with 2% sodium bicarbonate] 0.1N hydrochloric acid, and water. The product was then chromatographed on Khatman KO. 1 paper in the toluene-propylene glycol system of Zaffaroni and Burton (9). The cortisone acetate was located by an ultraviolet scanner and the spot eluted with ethyl alcohol. The cortisone acetate was taken up in ethyl acetate and washed with water to remove traces of propylene glycol and was then rechromatographed on K h a t man No. 4 paper using a benzenemethanol-water (4 parts of benzene, 2 parts of methanol] 1 part of water) system (6). The spot was located, divided into upper and lower sections in an arbitrary manner, and eluted with alcohol, and the solvent filtered through sintered glass and made up in a convenient volume for estimation of steroid and radioactivity. Corticosteroid was determined in aliquots of these samples, carrier, and standard solution ( 7 ) . Appropriate aliquots of the samples were assayed for radioactivity. Let A = counts per minute in any arbitrary volume of solution of cortisone acetate. B = steroid content in this volume. C = carrier added (for these experiments C = total steroid present, as the small sample can be neglected). D = specific activity (c.p.m. per y) of acetic anhydride determined by acetylating 100 y of cortisone and purification as above without addition of carrier. X = sample in y . X = ACjED. When 100 y of cortisone were acetyl1430
ANALYTICAL CHEMISTRY
I.
Determination of 0.2 to y of Cortisone
3.0
Cortisone, y % Present Found Recovered 0.92 92.0 1.000 107.5 2.000 2.15 2.96 98.6 3.000 0.89 89.0 1.000 0.64 91.4 0.700 0.65 92.8 0.700 102.5 0.400 0.41 0.21 105,o 0.200 Mean 97.4
Table II.
Reproducibility of Analysis
of 0.063-7Samples of Cortisone Sample Cortisone in No. Original Sample, 1
0,055
0,058
2
0.058
3
0.058
0.062 0.045
4 5 6 c
0,073
0.070
0.064 0.067 0 072 0 068 0,054
0.054 8
0.053 0 063 Mean 0 061 f 0.0075 S.D. Upper and lower portions of each isolated chromatographic spot. Q
ated and purified by the above procedure] a specific activity of 8260 c.p.m. per y was obtained with the sample of acetic anhydride used. The data for the analysis of 0.2 to 3.0 y of cortisone are recorded in Table I. Analysis within 10% is practical. Further chromatography of several of these samples of cortisone acetate on ethyl alcohol-silica gel eluted with 1.5% ethyl alcohol in methylene chloride resulted in no cliange in specific activity.
The 0.05-y level appears to be the present limit of sensitivity for this analysis. The authors have no explanation for the blank value, and further chromatography would probably remove the extraneous counts from the carrier acetate and allow analysis of considerably smaller quantities. ANALYSIS O F MIXTURES
The authors’ interest in this procedure is its application to crude mixtures of steroids isolated from blood and tissues. As a model separation] the quantitative resolution of cortisone, corticosterone, dehydroepiandrosterone, and androsterone was studied a t submicrogram levels. The acetates of these substances were readily separated into the two corticosteroid derivatives and a mixture of the C-19 acetates by paper chromatography in the toluenepropylene glycol system. The mixture of dehydroepiandrosterone and androsterone acetates could not easily be separated] except by forming the epoxide of dehydroepiandrosterone by treating this mixture with perbenzoic acid. To the dried mixture of C-19 acetates in a small tube was added 0.2 ml. of 0.1M perbenzoic acid in chloroform. After standing overnight in the refrigerator, the reaction mixture was taken up in 5 ml. of ethyl acetate and washed with diluted sodium bicarbonate and water. The solvent was evaporated and the mixture applied to Whatman KO.3 paper. Dehydroepiandrosterone acetate epoxide and androsterone acetate were readily separated by development with petroleum ether-methanol-water, 100-8615. Spots were located by treating a 1-mm. strip of the chromatogram with alkaline m-dinitrobenzene, The calculation was the same as for cortisone. Specific activity of the reagent is evpressed as counts per minute per millimole for this calculation. Table I11 shows the results of the analvsis of two such mixtures. This degree of accuracy for the quantitative separation of steroids a t a submicrogram level is adequate for many studies. RECOVERY
REPRODUCIBILITY
OF
ANALYSIS
Into each of 11 small glass-stoppered test tubes were measured 100 pl. of an alcoholic solution of cortisone. Subsequent colorimetric analysis of the stock solution showed these samples t o contain 0.063 y of cortisone. These samples n-ere acetylated as described above. After evaporation of the reagents, 217.5 y of nonradioactive cortisone acetate mere added. Purification and analysis were carried out as for the larger quantities of cortisone. Test tubes containing only acetic anhydride and pyridine mere worked up in a similar manner and an average blank value of 0.0184 y of cortisone was obtained. The results in Tahle I1 are corrected for this blank.
OF
EXCESS REAGENT
When large numbers of analyses are contemplated] it is worth while to recover the radioactivity for reconversion to acetic anhydride. The sulfuric acid used for absorption of excess reagent is poured into ice water. This mixture is then steamdistilled. The distillate is brought t o pH 9 with sodium hydroxide and distilled to a small volume. The sodium acetate is brought to dryness with a stream of nitrogen and dehydrated as described above. RECOVERY O F ADDED STEROIDS FROM PLASMA
Two milliliter aliquots of heparinized
human Dlasn~awere diluted a-ith 10-ml. samples of isotonic saline, which contained either no steroid or a mixture of 0.2 y of hydrocortisone and 0.1 y of corticosterone. The diluted plasmas were extracted three times with half volumes of chloroform. The combined chloroform extracts were washed with 0.5 volume of 0.1N sodium hydroxide and twice with o,5 of water. The chloroform extracts were then el-aporated by means of a gentle stream of air and the residues taken up in 20 ml. of petroleum ether. The petroleum ether was extracted three times with 20 ml. of 70% methanol. The methanol eltracts were combined and evaporated under the air blast. The residues were then taken up in alcohol and transferred to small test tubes. Following removal of the alcohol by evaporation, the samples were allowed t o d r y in a vacuum desiccator overnight. The samples were then acetylated and worked up exactly as described above for mixture of hydrocortisone and corticosterone. Separate plasma pools were employed for the column and chromatogram studv. The results of recovery studies are shown in Table IV. The chloroform-extracted plasma was diluted with 100 ml. of saline and brought to pH 1.0 with sulfuric acid to hydrolyze the conjugated plasma ketosteroids. Androsterone and dehvdroicoandrosterone were added to half the samples. The samples a,ere continuously extracted with ether for $8 hours and after removal of ether the residues were subjected t o petroleum ether70% alcohol distribution, a c e t h t i o n , and separation as described ahoue.
-411this even after repeated chromatography, gave results entirely too high and completely nonreproducible* It was that Of thcse compounds from plasma require preliminary chromatography before acetylation. DISCUSSION
The isotopic derivative technique n ould appear to be practical for separation and estimation of fairly pure altollolic steroids a t the submicrogram level. The major problem for each analytic separation is attainment of radiopurity of the derivative. The extent of this problem depends on the complexity and nature of the Sample. Analysis of crude extracts is much more difficult. I n the work reported
Table 111. Substance
Analysis of a Model Mixture by Acetylation Method
In SamHydrocortisone Corticosterone Dehydroepiandrosterone Table IV. so
Recovery of Hydrocortisone and Corticosterone from 2 MI. of Plasma
HJ,drocortlsone, Xdded
0
2
3 4 3
2 5
-
6 I
pie, Y 0 050 0 100 0 200 2 000
Mixture -4 Mixture B Found, % of In Sam- Found, Liof y Theory ple, y y Theory 0 067 134 0 100 0 092 92 0 117 117 0 100 0 080 80 0 320 160 0 100 0 082 82 2 20 110 0.250 0 246 98
0 0 0 020
0 020 0 020 Column 0 0 0
0 0 0 2 0 2
Coi ticosteione, y Recovery -1dded Found y Chromatogram Technique. Plasma Sample A 0 027 0 0 065 0 026 0 0 041 0 027 0 10 0 123 0 070 70 0 055 0 028 140 0 10 0 159 0 106 106 0 053 0 026 130 0 10 0 143 0 090 90 0 061 0 034 170 0 0 055 Technique Plasma Sample B Silica Gel Column Technique 0 17 0 0 124 0 18 0 0 105 0 17 0 1 0 245 0 126 1213 0 1 0 240 0 121 121 0 15 0 1 0 210 0 091 91 0 31 0 15 75 0 0 110 0 36 0 20 100 0 0 123 Found
Recovery
Y
here, adequate recovery for this sample level was obtained for hydrocortisone and corticosterone but suitable analyses were not possible for androsterone or dehydroepiandrosterone. This report is not intended to emphasize the absolute values obtained for hydrocortisone and corticosterone for peripheral blood, but only the recovery of added steroid. The procedure is required in this laboratory to detect differences from a base line level. If absolute ralues were required, repeated fractionation by a variety of procedures to establish radiopurity would be in order. When acetylation is performed on plasma extracts, a large number of derivatives are formed which distribute themselves along the chromatogram. Therefore several steraids OCCUPY the same Position on successive chromatograms. If absolute values rather than differences are desired, it would be appropriate to convert the acetylated compound to a suitable derivative which retained the acetate group, in order to assure adequate separation.
-~
oc
LC
LITERATURE CITED
(1) hvlvi, p., Simpson, s -4, ‘hit, J. F., Whitehead, J. K., Proc. b n d Radzo?sotope Conf., Oxford I, M e d . and Physzol. Applzcatzons, 313 (1954). (2) ~ ~ ~n,L., l Federatzon i ~ ~ proc, ~ 15, , No. 1,219 (1956). (3) Bojesen, E., Scand J . Clzn. &- Lab. Inbest 8, S o 1,55 (1956). (4) Bojesen, E., Keston, 4.,Carsiotis, II., -4bstracts of Comm. XIX, International Phyiiological Congress, Montreal, p. 220, 1953. (5) Bush, I E I Bzochem J . 5 0 , 9 0 3, 370 (1951). (6) Hickinbottom, IT. S., “Reactions of Organic Compounds,” p. 232, Longmans Green, London, 1936. (i)
j
p.j
Ellis, J. P., Proc. SOC.Exptl. Bzol. and M e d . 88, 643 (1955). (8) Keston, A s , Udenfriend, s., Cannan, R . K , J . A m Chem. Soc. 71, 249 (1949) (9) Zaffaroni, A , , Burton, R. B , J Bzol. Cheiiz. 193, 769 (1951) RECEIT ED for revieir Sovember 19, 1957. -iccepted February 24, 1958 R ~ search supported in whole bv the U S. Force under Contract No. AF 41(657)-34, monitored by the School of Aviation l\Iediclne, ~ ~ ~ ilr d Force ~ l Base, Tex.
VOL. 30, NO. 8, AUGUST 1958
1431
~
h
