Purificution of Urinary 17-Ketosteroid Extracts f o r Inf ra red An a Iys is W. ROY SLAUNWHITE, Jr., and LAVALLE NEELY Roswell Park Memorial Institute, Buffalo, N. Y.
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The method commonly employed for preparing neutral urinary extracts (containing 1 -/-ketosteroids), followed b y gradient elution chromatography, has produced fractions which frequently have yielded infrared spectra difficult to identify with assurance. Washing the concentrated urinary extracts with large volumes of alkali and acid, followed b y treatment with vegetable charcoal, eliminates so much of the contaminating material that the fractions produced b y gradient elution chromatography give infrared spectra nearly identical with those o f the standards.
A
of normal male urine mas hydrolyzed, extracted, and divided into three portions in order to test the effect of washing with acid and alkali and of treatment with charcoal. Six liters of urine was hydrolyzed by refluxing for 10 minutes with 900 ml. of concentrated hydrochloric acid. After extraction of the 17-ketosteroids with methylene chloride, the solvent was evaporated t o 90 ml. The extract was then divided into three equal portionsA, B, and C. Each was treated as described beloly. POOL
Sample A was washed three times with equal volumes of 1N sodium hydroxide and once with an equal volume of water. The solvent was evaporated on a steam bath under a current of nitrogen. Sample B was treated in the same way. The residue was then dissolved in 25 ml. of chloroform and 50 mg. of vegetable charcoal (Darco, obtained from the Coleman and Bell Co.) was added. After standing at room temperature for 10 minutes, the charcoal was removed by filtration through Whatman No. 1 filter paper and was washed with 75 ml. of chloroform. The solvent was evaporated in the same way as Sample A. Sample C was washed in the same manner as Sample A. It was then washed three times with equal volumes of 1N hydrochloric acid and once with an equal volume of water. The solvent was evaporated and the residue treated with charcoal in the same fashion as Sample B. Total 17-ketosteroids were deter1614
ANALYTICAL CHEMISTRY
I
1800
~
600
I
~
1400
/
1200
iobo
I
860
WAVE NUMBERS IN CM.-1 Figure 1. Infrared spectra of Fraction 11 from gradient elution column
I . Extract washed three times with 0.1 volume of alkali (480 y of 17-KS) A . Washed three times with equal volume of alkali (800 -!of 17-KS) B. Washed as in -4,then treated with charcoal (700 y of 17-KS) C. Washed as in A , then washed three times with equal volume of HC1, then treated with charcoal (750 y of 17-KS) STD. Pure androsterone (700 y )
mined by the method of Klendshoj (6), except that color development was allowed to proceed for 1 hour in the refrigerator instead of at room temperature and absorbances were measured a t 440, 520, and 600 ml.r (3). Each sample was chromatographed by the gradient elution method of Lakshmanan and Lieberman (7); alternate fractions from the fraction collector were analyzed for their content of 17ketosteroids. Infrared analysis was performed on a Baird double-beam infrared spectrophotometer by the potassium bromide
pellet technique (1). Potassium bromide (Mallinckrodt or Fisher analytical reagent grade) was recrystallized once from water and dried a t 150" C. for 48 hours. It was finely ground in a porcelain mortar, and that portion which passed through a 230-mesh sieve was again dried a t 70" to 80' C. for 12 hours and bottled. The 17-ketosteroids contained in one or more vials from the fraction collector were dissolved in about 0.2 ml. of acetone and transferred by pipet to a 1-ml. beaker containing about 2.5 mg. of potassium bromide powder (order of addition is not important).
Table I. SO.
... I" I1 I11 IV
V
Urinary
17-KS Values
before and after Chromatography
Fraction Same A Total Zimmerman chromogens applied to column 12.8 Total 17-KS identified 7.8 Dehydroepiandrosterone 0.15 Androsterone 2.80 Etiocholanolone 4.54 11-Iietoetiocholanolone 0.09 llp-Hydroxyetiocholanolone
E
Sample, X g . C
12.7 7.5
8.1
0.14
3.34 3.61
0.17
0.21
0.27
9.0
0.21 2.48
5.03 0.13 0.27
-4v. i. u 7.8 2 ' 0 . 3 0.17 1 0 . 0 3 8 2 . 8 i rt 0.44 4.39 i 0.73 0 . 1 3 i 0,040 0.26 C 0.017
Sample A washed with NaOH, B with NaOH and treated with charcoal, C with SaOH and HC1 and treated with charcoal.
The acetone was evaporated a t 70" to 80" C. and the potassium bromide powder transferred to a '/,-inch die (Baird Associates, Inc., Cambridge, Mass.). The die was placed in a vacuum chamber assembly (Baird Associates, Inc.) which was evacuated with
an oil pump for 2 to 3 minutes. The pellet was then formed by applying a pressure of 4000 pounds per square inch for 3 minutes. The clear 0.25 X 0.031 inch pellet was inserted in a beam condensing unit (Baird Associates, Inc.) which, in turn, was placed in the sample
-,
J
s
BOO
1600
1400
1200
io00
aoo
WAVE NUMBERS IN CM;' Figure 2. Infrared spectra of fraction IV from gradient elution column
I.
Extract washed three times with 0.1 volume of alkali (500 y
.4.
of 17-KS)
Washed three times with equal volume of alkali (400 y of 17-KS) B. Washed as in A , then treated with charcoal (500 y of 17-KS) C. Washed as in A , then washed three times with equal volume of HCI, then treated with charcoal (500 y of 17-KS) STD. Pure 11-ketoetiocholanolone(500 7 )
beam. A compensating plate to offsct the reflectance losses of the two lenscs was inserted in the reference beam. Recovery experiments \\ere performed with varying amounts of dehydroepiandrosterone and Il-ketoetiocholanolone; three types of charcoal. Darco, Sorit -4,and bone; and two solvents, chloroform and ether. In each case, 2 mg. of charcoal was added to the steroid dissolved in 2 ml. of solvent and allowed to stand for 3 minutes a t room temperature. The charcoal was removed by filtration and washed with 10 ml. of solvent. The solvent was removed and 17-ketosteroids were determined. RESULTS AND DISCUSSION
The urinary 17-ketosteroid values (Zimmerniann chromogens) obtained before and after chromatography are given in Table I. Treatment with decolorizing charcoal had little effect upon the total 17-ketosteroid values before chromatography (Sample B L'S. Sample A), presumbly because measurement a t three wave lengths had canceled out the absorption due to urinary pigments. When the charcoal used in purification of Sample B was eluted with methanol, pigments were obtained which produced a bronm color in the Zinimermann reaction. The hydrochloric acid n-ashing of Sample C. on the other hand, extracted a yelloly fluorescing material which was, in turn, extractable from the neutralized solution with ether. The broJvnish yellow residue left after evaporation of the ether was largely responsible for the difference in 17-ketosteroid value between B and C before chroniatography. An infrared spectrum of this impure material revealed no absorption in the carbonyl region (1660 to 1780 cm.-I) or hydroxyl region (3200 to 3600 em.+); the adsorption in the fingerprint region was not typical of a steroid. After chromatography the amounts of steroid identified in all three samples agreed well with each other and with the original value for Sample C. The average value of total steroids identified (Table I) was 7.8 mg., which is 87% of the initial value (9 mg.) of Sample C, the most highly purified extract. Unidentified chromogens were invariably eluted in the first two tubes and in an early peak preceding fraction I. The identity of the five major 17ketosteroid fractions resolved on the gradient elution column is shown in Table I for each of the three methods of treatment. Quantitatively the column gave fairly reproducible results regardless of the type of purification. Qualitatively, however, the type of purification had a marked effect on the quality of the infrared spectra, as can be seen in Figures 1 and 2. As the results for each fraction were similar, only the VOL. 29, NO. 1 1 , NOVEMBER 1957
1615
spectra of fraction I1 (androsterone) and IV (11-ketoethiocholanolone)are shown. For comparison, the spectra of pure standard and of a sample obtained from an extract washed with small volume) volumes of alkali (3 X with are shown. A steady improvement in the spectra is noted. However, the amount of purification is not the same in all cases, for spectrum C of androsterone (Figure 1) more closely approximated that of the standard than did spectrum C of 11-ketoetiocholanolone. In both cases, however, the final spectra (spectra C) were sufficiently detailed for even an unpracticed eye to identify with assurance. The amounts of steroid used in obtaining the spectra of Figure 1 were three to five times the amounts used in routine identification. Ordinarily 100 to 200 pg. are used. Two steroids, 11-keto- and lla-hydroxyandrosterone, normally present in urinary extracts in small quantities, as well as numerous artifacts, such as A2-androsten-17-one, A3,5-androstadien17-one, 3~-chloro-A5-androsten-17-one, Ag(11)-etiocholen-3a-ol-17-one, and A9(I1)androsten-3a-ol-17-one, were not identified because of technical difficulties. The gradient elution technique used does not separate compounds differing by only one isolated double bond. For example, epiandrosterone and dehydroepiandrosterone, androsterone and Ag(11)-androsten-3a-01-17-one, and etiocholanolone and A9(11)-etiocholen-3a-ol17-one are eluted in fractions I, 11, and 111, respectively. In addition, an isolated double bond in the 9 (11) position does not absorb appreciably in the infrared region (6) nor does it greatly influence the spectrum of the rest of the molecule [cf. spectra 144 and 148 (41. The unsaturated compounds may be separated from their saturated analogs by treatment with perbenzoic acid followed by paper chromatography (IO) or they may be determined spectrophotometrically in sulfuric acid solution without separation ( 2 ) . 11P-Hydroxyandrosterone is usually not resolved from 11ketoetiocholanolone, but appears in the trailing edge of fraction IV. If present in sufficient quantities, it is easily detected by infrared spectroscopy. The effects of three types of charcoal and two solvents are shown in Table 11. It is readily apparent that Darco charcoal was less absorptive than the other two and that while there was no difference between solvents as far as 11-ketoetiocholanolone was concerned, recoveries were lower with dehydroepiandrosterone when ether was used as a solvent. Methylene chloride was equivalent to chloroform (data not shown). The recoveries, where ether and Norit A were used, are in agreement with those of Pincus and Pearlman (9), who used the same reagents on urinary extracts. Lombardo and associates (8) 1616
ANALYTICAL CHEMISTRY
Table II.
Effect of Type of Charcoal and Solvent on Recoveryof Dehydroepiandrosterone (DHA) and 1 1 -Ketoetiocholanolone (1 1 -KE)
Norit A Steroid Amt., y CHClr Et20 DHA 50 7 8 f 1 . 7 63 f 2 . 0 75 8 8 z k 4 0 8 1 f 1 6 100 8 3 f 2 6 7 4 A 4 7 11-KE 50 77 f 3 . 0 77 f 2 . 1 68 7 4 f 3 . 1 74 f 3 . 0 136 82312.1 84zk2.3 4
% Recoverys Type of Charcoal Bone CHCls EtpO 82 1 2 . 6 75 f 1 6 9 3 f 3 8 83zk16 8 9 f l O 86+16 81 f 1 . 6 82 h 5 . 9 82 f 3 . 2 81 f 0 . 7 8 5 f 2 . 6 88 zk3.8
Darco CHCls EtzO 89 f 1 . 2 82 f 3 . 0 97f20 91f26 97fl7 90f47 8 6 f 1 . 0 86 A 4 . 0 9 4 f 1 . 6 93 f 2 . 5 95 A 2 . 0 9 1 h 3 . 6
Average and estimate of standard deviation of 3 determinations.
also obtained good recoveries of both 17-ketosteroids and corticosteroids using 60% ethanolic benzene and Mallinckrodt activated charcoal. Table I1 also shows that the ratio of the weight of charcoal to steroid is important. When the ratio was 40, losses were significantly greater than when the ratio was 20 or less. When Darco charcoal was used a t a ratio of 20 with chloroform as a solvent, recoveries were nearly quantitative. In one instance substantial loss of 17ketosteroid occurred a t high charcoalsteroid ratios. This was a sample weighing 26.6 mg. and containing 14.7 mg. of carbon-14-labeled etiocholanolone. ilt a 20 to 1 ratio there was a 50% loss of 17-ketosteroid, a t 5 to 1 ft 35y0 loss, and a t 2 to 1 a 10% loss. However, when the impurities greatly exceeded the 17-ketosteroid present, as in the urinary extracts, no loss of etiocholanolone occurred (Table I). The ratio of charcoal to steroid in the urinary extracts was 6. Although it is not documented here, time of exposure of the steroid to the charcoal is important-i.e., long exposure will result in losses. The 10-minute exposure allowed for the urinary extracts is the maximum permitted without loss of steroid; 3 to 5 minutes are required for absorption of urinary pigment. Time and temperature, of course, have a reciprocal relationship to each other-i.e., in boiling chloroform the time of exposure to charcoal should not exceed 3 minutes. The effect of treatment with charcoal on the total 17-ketosteroids was determined on 27 daily specimens from six patients. The neutral urinary extracts were treated fn the same manner as Sample C, except that 17-ketosteroid was determined before treatment with charcoal as well as after. Values ranged from a 35% decrease t o a 52Q/, increase as compared to the values before treatment. In many instances the increase was due to elimination of material producing a brown color in the Zimmermann reaction. The average
of all 27 determinations was a decrease of 7.8%. CONCLUSIONS
Substantial purification of neutral urinary extracts can be achieved by washing the extract dissolved in a small volume of methylene chloride exhaustively with I N sodium hydroxide and then with 1N hydrochloric acid and by treatment with Darco vegetable charcoal for 5 to 10 minutes a t room temperature. The ratio of charcoal to steroid by weight must be less than 20. For an extract of a normal 24-hour urine specimen, 50 mg. of charcoal is recommended. The conditions for treatment with charcoal must be observed strictly; other-rise, considerable loss of steroid may occur. Because the variability among different batches of Darco vegetable charcoal is not yet known, each new batch should be tested for recovery of added 17-ketosterids. LITERATURE CITED
Anderson, D. H., Woodall, 3. B., AKAL.CHEM.25, 1906 (1953). Bitman, J., Rosselet, J. P., Reddy, A. &I., Lieberman, S., J . Biol. Chem. 225, 39 (1987). Chang, E., Slaunwhite, W. R., Jr., J . Clin.Endocrinol. and Metabolism 15, 767 (1955). Dobriner, K., Katzenellenbogen, E. R., Jones, R. S . ,Infrared Absorption Spectra of Steroids,” Interscience, New York, 1953. Jones, R. N., Humphries, P., Packard, E., Dobriner, K., J . Am. Chem. SOC.72,86 (1950). Klendshoj, 2;. C., Feldstein, M., Sprague, A, J . Clin. Endocrinol. and Metabolzsm 13,922 (1953). Lakshmanan, T. K., Lieberman, S., Arch. Biochem. Biovhvs. 53. 258 (1954). (8) Lombardo, M. E., Viscelli, T. A , Mittelman, A., Hudson, P. B., J . Biol. Chem. 212,353 (1955). (9) Pincus, G., Pearlman, W. H., Endocrinology 29, 434 (1941). (10) Rubin, B. L., Dorfman, R. I.> Pincus, G., J . Biol. Chem. 203, 629 (1953). .
I
RECEIVED for review November 21, 1956. Accepted June 12, 1957.
