1853
V O L U M E 28, NO. 12, D E C E M B E R 1 9 5 6
(6) Crouthamel, C. E., Johnson, C. E., U. S.Atomic Energy ('ommission, ANL-4924 (1952). (7) Hine, G. J., .VucZeonics 1 1 , S o . 10, 68-9 (1953). (8) Kahn, B.. Lyon, W. S., Ibid., 1 1 , No. 11, 61-3 (1953). (9) Lazar, S . H., Davis, R. C., Bell, P. R., Ibid., 14, No. 4, 52-3
tion rates. T h e application of the method continues to increase as complete gamma spectrometer systems are becoming commercially available and specific applications appear more frequently in the literature.
(10) LITERATURE CITED
(11) (1) AAIbert,€1. D., Rcc. Sci. Instr. 24, 1096-101 (1953). (2) Bell, P. R., "Beta and Gamma Ray Spectroscopy," li. Sieghahn. ed.. Interscience. S e w York. 1955. (3) Berger, .\I. J., Doggett, J., Ret. Sci. Iiiistr. 27, 269-io (1956). (4) Connally, R. E., I . R . E . Trans. NS-3, T o . 2 . 28-31 (1956). (5) Connally, 11, E., Leboeuf, .\I. B., .kS.kL. CHEM. 25, 1095-100 (1953).
(12) (13)
(1956). Lefevre, H. IT., private communication, General Electric Co., Richland, Wash. McIsaac, L-. D., V. S.Naval Radiological Defense Laboratory, TR-72 (1956). Miller, D . G., U. 8. Atomic Energy Commission, HW-39969 (1956). Morrison, G. H., Cosgrove, J. F., -&x.IL. CHEU. 27, 810-13 (1955).
R E C E I V E for D re\,iew July 10, 1956.
.Iccepted Septeinber 2 2 , 1930.
Ninth Annual Sommer Symposium-Analytical Problems in Biological Systems
Paper Chromatography in Steroid Determination LESTER M. REINEKE Research Laboratories, The Upjohn Co., Kalamazoo,
Mich.
Paper chromatograph? has been applied to the identification and quantitatite determination of steroids obtained from microbiological and chemical transformations. 4 clear indication of the probable structure is obtained b? ultraliolet absorption, chemical tests, and mobilit? (as compared to various known steroids) in a variet? of sollent s?stems of both the Bush and Zaffaroni t?pes. 4 quantitatile procedure using light absorption at 224 and 242 mp has been deleloped for progesterone and lla-hjdrox?progesterone. This procedure eliminates the necessit? for running blanks and steroid standards with each determination.
T
HE technique of paper chromatograph? is discussed here,
as applied to steroidal transformations induced microbiologicalli or chemically. The compounds considered are more polar than the sterols. I n these laboratories, paper chromatography is used only for identification or determination of purity, but the infoimation is also helpful in proof of structure. \T-hen Zaffaroni, Burton, and Keutman (31) reported the formamide-benzene and propylene glycol-toluene systems, the first practical method of applying paper chromatography to steroids brcnme available. [ I n this discussion, any solvent system (1-4, 1 1 , 16-17, 21, 23-25, 27, 28, 30) using a paper impregnated n i t h a high boiling polar solvent as the stationary phase and a nonpolar solvent saturated n i t h the stationarv phase as the mobile phase is referred to as the Zaffaroni type.] Later, Bush (6)used a series of s~ stems in n hich the stationary phase, water and methanol, is preferentially adsorbed onto the paper from the vapor of both phases during equilibration. The mobile phase, consisting of various nonpolar solvents, is used for development. With the Bush systems, a slightly elevated temperature is usually desirable. [Any solvent s>stem of similar nature (6, 8,14, 18, 19) is referred to here as of the Bush type.] Various systems for paper chromatography of steroids, other than sterols (5, 9, 13, 22, 26, 29), have been published but, in general, they are one of the above two types COMPARISOY OF DEVELOPRlEhT SYSTEMS
1Iuch larger quantities of steroid can be applied to the Zaffaroni type of chromatogram than can satisfactorily be applied to the Bush type. The development time of the former is generally longer, but in the Znffaroni type of system resolution of steroids having ultraviolet absorption can be conveniently followed by
removing the strip from the chamber for ult'raviolet' scanning. The sheet may then be dipped t'hrough the mobile phase and the development continued. This is not possible with the BiiPh type of system. I n order to speed up and sharpen the resolution, it is a common to reduce the amount of practice, first reported by Zaffaroni (28), stationary phase by dipping the filter paper through the stationary phase diluted with a volatile solvent such as methanol, The diluent is then evaporated from the paper chromatogram before the development is started. T n o modifications of Zaffaroni's procedures (28, 31) have been made. (1) S o wick is used to saturat'e the battery jar; the nonpolar solvents are sufficiently volatile to saturate the chamber rapidly. (2) Channeled sheets are not used, because they present greater manipulation problems, particularly when a sheet may be removed from the bath several times for observation. The slight sideways diffusion of the steroidal material is useful in the separation of a small quantity of material from a larger amount of a closely moving material. If confined to the narrotv channel, the "edge effect" can obliterate the space between compounds of similar mobility. The mobilities on these systems are expressed as a ratio to a standard steroid, Rs, because the solvent front has often moved off the chromatogram before sufficient' resolution has been ob-
Table I. Zaffaroni-Type Solvent Systems0 LiteraDried ture at System ReferPhase 370 C., Symbol ence Mobile Stationary Min. Cll (16) Methylcyclohexane Carbitol b .. CNF hlethylcyclohexane Carhitol" 10 PT (3;) Toluene Propylene glycol , . PTF (28) Toluened Propvlene glycolc 10 K-1 (1I ) CyclohexanePropklene glycolc 10 benzene (1 : 1) FBF (28) Benzene FormamideC 15 .. Skellysolve B e CFS Carbitol15 formamidef a Best grade of solvents commercially available is used. Only formamide is redistilled. Room temperature, 2 5 O ?C 2O C. * Diethylene glycol monoethyl ether. Diluted with methanol (1 : 1). Methanol evaporates during indicated drying time. d For materials such as 3-ketohisnor-4-cholenic acid, 27, glacial acetic acid can he added to reduce streakiness. e Essentially a normal hexane, b.p. 60-70' C,. f Two parts of Carbitol-formamide (1:l) dlluted with 1 part of methanol.
'
1854
*
ANALYTICAL CHEMISTRY
tained. An Rs difference of a t least 5% is generally needed for positive differentiation-e.g., two materials with Rs values (as of compared to lTa,2l-dihydroxy-4-pregnene-3,11,20-trione) 0.10 and 0.11, a 10% difference, could be easily separated from each other a i t h a sufficient development period. With Rs values of 2.0 and 2.1 (5% difference), the separation is more difficult. The Bush systems have an appreciably faster development time after equilibration. The resolution of the steroids is usually achieved with the advancing solvent front still on the paper. Hence, data can easily be expressed in terms of R f (relative mobility of the material to the solvent front). However, if the solvent front has been allowed to move off the chromatogram, the R f can easily be calculated by comparing the unknown to a control of knon-n value. An R , difference of as little as 0.02 is often sufficient for adequate resolution. No one system does a perfect job; hoaever, each system does a better job on one group of steroids than on another. For example, steroids such as 1la, lia,21-trihydroxy-4pregnene3,20-dione, 6P,lia,21-trihydroxy-4-pregnene-3,20-dione, and 1lp, 17~u,21-trihydroxy-4-pregnene-3,20-dione, which might require 10 days of development for adequate differentiation p i t h the PTF system, mag be differentiated in 3 hours of development time (after equilibration) n-ith the Bush B-5 system. On the other hand, 6p,17j3-dihydroxy-4androsten-3-one and 14a,17pdihydroxy-4androsten-3-one, are not separated by the latter system, but differentiate nicely with the P T F system in 3 days. I n practice, the results from several solvent systems are compared. No matter what type of system is employed, the use of a slower system is indicated if a steroid is moving too rapidly. If the material is moving too slowly, time may be saved by running another aliquot from the sample on a faster system. S o attempt is made to maintain exact values, as it has been found in these laboratories that mobility values can vary by +lo%. I n addition, nonsteroidal materials in the fermentation extracts enhance or slow the mobility of the steroidal material. It is essential to run the sample and mixture of suitable controls side by side as well as mixed together, until sufficient experience has been obtained for a particular organism on a given substrate. There is, in general, an inverse relationship betneen the mobility and the expected polarity of the steroid. To facilitate the resolutions of closely moving steroids, it has proved desirable to use a t least one chamber, 36 inches high, for each of the major development systems. Very frequently, a resolution is achieved on a 34-inch chromatogram which was unsatisfactory on a conventional 21- or 23-inch chromatogram. Often, the longer sheet has made it possible to obtain a complete picture of a bioconversion from a single chromatogram instead of two. Both Eaton and Dikeman No. 613 and Whatman No. 1 filter papers have been used. T h e former is preferred because it has superior wet strength when treated with Tollens reagent (31). Preivashing the filter paper is unnecessary for this Tyork.
Table 11. Bush-Type Solvent S>stems"sb System Composition il Skellysolve methanol, water-5:4: 1 B-1 Toluene, Skellysolve C, methanol, water-5 : 5 :7 :3 B-3 Benzene, Skellysolve C, methanol, water-333: 667: 800: 200 B-5 Benzene, methanol, water-2: 1: 1 See (6). b Best grade commercial solvents; temperature of 34' =t1O C. used for these systems. Essentially a saturated hydrocarbon fraction, b.p. 86-100' C.
sulfuric acid (0.3 ml.) is added and the flask is heated on a steam bath until the reagent is in solution. It is then diluted to 200 ml. with 3-A ethyl alcohol. This reagent was independently developed in this laboratory in cooperation with B. J. Magerlein. Kochakian and Stidworthy ( 1 1 ) have developed a similar reagent using hydrochloric acid. 2,4Dinitrophenylhydrazine has proved useful for the detection of ketones a t C-3 or C-20, but does not react n i t h those a t C-11. Steroids with a conjugated ketone appear orange, while the other ketosteroids appear as yellow spots against a light yellow background. This test is sensitive to about 8 y . The 20-ketone is less reactive than the 3-ketone, and a 3-keto-l,4diene is still less reactive and in some cases is not detected a t a level of 32 y . This order of reactivity is in keeping with the findings from chemical syntheses. When this reagent is followed with Tollens reagent, a more stable chromatogram is obtained. Steroids containing ketones now appear as yellow spots against a gray background. If the steroid has an a-keto1 side chain as well as a 3-ketone, the spots are a dark yellow-gray. When no 3-ketone is available, the keto1 side chain produces a dark gray to black spot against the lighter gray background. Apparently, a 21-hydroxyl or acetoxy group offers sufficient hindrance so that the 20-ketone will not react readily with the dinitrophenylhydrazine reagent. Tollens reagent can be used by itself. Besides the cortical side chain, it has been found useful in detecting steroids such as 4p-bromo-17a-hydroxy-5p-pregnane-3,11,20-trione. A sensitivity of 1 y is often obtained. Kritchevsky and Kirk (12) developed a phosphomolybdic acid reagent 1% hich generally detects steroidal hydroxyl groups. The sheet is dipped in a 10% (w./v.) phosphomolybdic acid (Fisher 6-237, Fisher Scientific Co.) in absolute methanol and then heated a t 90" C. for 5 minutes. The spots appear bluish to black on a green background. Sixteen micrograms is generally necessary for detection. This method does not work well with sheets which have been treated with formamide as the stationary phase. Nor can this reagent be used satisfactorily on a sheet treated with the test reagents previously discussed. Because of its low sensitivity, it is used only when the other tests give poor detection. MOBILITY RELATIONSHIPS
METHODS OF DETECTION
The ultraviolet absorption of the conjugated ketone in the A ring is the most useful identification test for the steroids encountered in this program. The scanner of Drake and associates ( 7 ) of these laboratories shows the materials as dark spots against a fluorescent background. By using a suitable photographic paper (Kodagraph Contact Standard) placed directly against the chromatogram, a full size photograph may be made as a permanent record and for convenient analysis of results. This leaves the paper available for color tests if desired. For ketosteroids that do not have ultraviolet. absorption, a spray reagent of 2,4-dinitrophenylhydrazine was developed. The solution is prepared by suspending 300 mg. of 2,4-dinitrophenylhydrazine (Eastman 1866) in 5 t o 6 ml. of 3-8 ethyl alcohol (U. S. Industrial Chemicals, Inc., SDA). Concentrated
Several studies ($3, 27) on the relationship of mobility to chemical structure have been reported. A4number of exceptions occur, not explicable from the author's experience. These reversals actually aid in making the probable identification more accurate. For example, 6p-hydroxy-4-pregnene-3,20-dionemoves more slowly than 14a-hydroxy-4-pregnene-3,20-dioneon all systems that have been tried, except the Bush A. On the CFS system, 5a-pregnane-3,11,20-trione moves faster than 5p-pregnane3,11,20-trione. This is contrary to the usual experience that the 5a-steroids move more sloir.1y. For example, a separation of 11a-hydroxy&~-pregnane-3,20-dione from 1la-hydroxy-5ppregnane-3,20-dione, the latter compound has the greater mobility-that is, is less polar. Both Savard (23) and Zaffaroni (27) have generalized that the 5a-configuration is the less polar, in contrast to the present author's findings.
V O L U M E 2 8 , N O , 12, D E C E M B E R 1 9 5 6 The products of a microbiological conversion of a steroid can be very complex-e.g., a Cal steroid may have the side chain removed, the D ring altered, and a hydroxyl group added (20). A compilation of the mobilities of a number of steroids in several different development, systems together with the method of detection is very useful in establishing the probable identification of the steroids in samples submitted for analysis. A common reference for the mobilities facilitates the identification of such varied products. A study of Table I11 shows that the faster moving members of one series-for example, the C19steroids-overlap with the s l o w r members of another, the C21 steroids. This is typical of the results obtained. A difference in reaction to a test is as useful as a different solvent system. For example, lTa,2Op,21-trihydroxy-4-pregnen3-one and 17~,21-dihydroxy-4-pregnene-3,11,20-trione are difficult to separate on the PTF system, but t'he lat'ter compound gives a positive Tollens test. 17P-Hydroxy-4-androsten-3-one and 21-hydroxy--l-pregnene-3,20-dione have identical mobilities on the Bush B-3 system. Again, Tollens reagent would quickly show the difference. Because it is generally desired that new steroids be isolated on a macro scale, characterization of an unknown by paper chromatography of derivat'ives obtained by micro techniques, a method used by Zaffaroni and Burton (27, 28) and others, is not attempted. Rather, a variety of solvent systems are relied upon to limit the number of steroid possibilities. As many as 10 or 15 steroids may move together when a single system is employed. When several systcms are used, there are very few steroids which are not differentiated from each other if they are a t least as polar as 11a-hydrosy-?$-pregnane-3,20-dione. To help identify a steroid, physical and chemical properties are compared n-ith those of as many steroids as possible. The characteristics compared are mobilities on several solvent systems, ultraviolet absorption, and functional groups as indicated by chemical reagents applied directly to the developed chromatogram. Table 111 gives this pertinent information for 121 steroids. I n this table the steroids are related to the solvent front or to the same standard steroid Tyithin a given development system. The values are obtained by direct comparison to key steroids for which average values from several determinations are used. To facilitate the location of a steroid, Table 111 is divided into the following sections.
A. Steroids of 19 carbon atoms in the basic molecule with a carbonyl group at position 17. 17p-Hydroxj4-androsten-3-one is also included in this sect,ion for comparison purposes B. Steroids of 19 carbons with a hydroxyl group in the 17pposition C. Steroids of 18 carbon atoms (19-norsteroids) D. Steroids of 21 carbon atoms, except those n i t h an a-keto1 or glycol side chain. For purposes of comparison -5th other mon0hydroxy-4-pregnene-3~2O-dione steroids, 21-hydroxy-4-pegnene-3,20-dione is also included E. Steroids of 21 carbon atoms with an a-keto1 or glycol side chain I n each division, the compounds are listed in order of increasing number of functional groups. Carbon-carbon double bonds are not included in the number of functional groups. Within anjcategory of total functional groups, the ones with the highest number of carbonyl groups are listed first; then they are listed by number of hydroxyl groups. The compounds n-ith the a-orientation are listed first, with the 3a-hydroxyl group arbitrarily listed ahead of a &-hydrogen in a saturated steroid. When the functional groups are the same, listing is by decreasing degree of saturation. SEMIQUANTITATIVE PROCEDURES
Visual Estimation. I n TTorking with conversion products formed by microorganisms, two assumptions are made: that the steroid is not destroyed by the organism, and that the steroid or
1855 its conversion product is completely extracted from the beer. Although these assumptions are often not true, the qualitative results obtained by comparing the spot n i t h the amount of the control give significant information in plotting the steroid balance hIore precise quantitative and semiquantitative procedures using paper chromatography can readily be developed. Often a semiquantitative visual estimation is entirely satisfactory and can be obtained in considerably less time. Because such estimations a t best will be 5 2 0 % of the amount present, the minor steroidal impurities are often estimated instead of the major transformation product. Levels of the suspected impurities comparable to that expected in the sample are used. For exestimated ample, crystalline 1la-hydrosy-4-pregnene-3,20-dione to contain 2 % 4-pregnene-3,20-dione is run a t levels of 200 and 400 y along n i t h levels of 4-,8-, and 16--/ quantities of 4-pregnene3,20-dione for visual comparison. If the material is fairly pure, it is assumed that the balance is the major steroidal component. For the impurities, the error may be i 50%. However, if the impurity is present to the extent of only 1 or 2%, the range is still very acceptable. The actual significance of the sample should be kept in mind. When this technique was applied to large scale fermentation studies, using the ChlF system, it was possible to give a good estimation of the residual 4-pregnene3,20-dione within 2 hours of sampling. Spectrophotometric Analysis. For more precise determination of a steroid having ultraviolet absorption, the material may be eluted from the developed chromatogram and estimated spectrophotometrically I t is necessary to correct the apparent absorption of the steroid for nonsteroidal material (blank) having ultraviolet end absorption, xdiich is also eluted from the paper. Complete removal of this latter material from the chromatogram is very difficult. Compeneation can be readily made for it, and the removal is not attempted. Experimentation showed that the ultraviolet absorption curves of the eluate of a number of blank control strips were similar in shape, although they varied considerably in intensity, depending upon the size of the sample. Thus, the approach of Johnson, Struck, Scott, and Stafford (10) of this laboratory could be used for handling a component of varying ultraviolet absorption intensity but of constant shape in a tlvo-component system. I t reduces the quantity of the blank to a numerical ratio. This greatly simplifies the conventional algebraic handling of spectrometric data for a tn-o-component system. For the blank, a ratio mas established of the abeorbance a t a point from the ultraviolet curve R here the blank showed good absorption (224 mp) and the absorbance a t a point where the steroids being used had good absorption (242 mp). The absorbance of a 1 mg. per ml. solution of the steroid a t the same wave lengths was also determined. The calculation is done as indicated in the follon ing formula:
'
'242
-
R X K?(?- Ii224
x
T
xP
= mg. in aliquots
where
R K V P D
= = = = =
ratio of D224/D242mp for the chromatogram blank absorbance of a 1 mg. per ml. solution of steroid volume of elution solvent pipet correction when micropipets are used (100/95) absorbance readings at 224 and 242 mp for the sample
The determination of lla-hydioxy-4-pregnene-3,20-dione and 4pregnene-3,2O-dione in a mixture will serve as an example.
Aicalibrated micropipet holding 0 01 ml. of solution containing 500 y is applied as a single spot on a propylene glycol-impregnated sheet, and the paper strip is developed with toluene overnight. The chromatogram is removed from the developing chamber and dried a t 105" C. for 2 hours. The area containing the steroid as seen on the scanner is outlined in pencil, and then cut from the developed chromatogram into a 250-ml. Erlenmeyer flask. Absolute methanol (50 ml.) is added and the flask is shaken on a rotary shaker for 2 hours. After the eluate is filtered through a coarse fritted funnel, an aliquot is read on a Beckman DU spectro-
1856
ANALYTICAL CHEMISTRY -
Table 111.
Steroid Mobilities
~
CRi,
5a-Androstane-3,17-dione 4-Androstene-3,li-dione 1,4-hndrostadiene-3,17-dione 4,9(1l)-Androstadiene-3.17-dione 3p-Hydroxy-5-androsten-17-0ne
a b
2.7
3p-Acetoxy-5-androsten-17-one 17B-Hydroxy-4-androsten-3-one Testololactone 4-Androstene-3,6,17-trione 4-Androstene-3,Il. 17-trione
d b b
Compound
A.
CISsteroids
c
b d
b
,
6p-Hydroxy-4-androst ene-3,17-dione 11a-Hvdroxv-4-androstene-3.17-dione
B.
~
Detection“
C I 17@-hydroxysteroids ~ 17~-Hydroxy-4-androsten-3-one 178-Hydroxv-l.4-androstadien-3-one 17p-.~cetoxy-4-androsten-3-one 17~-Propionoxy-4-androsten-3-one 17~-Cyclopentylpropionoxy-4-androsten-3-one
b b b b b b b C
b b b
Rs b
P TL
PTF,
. . .f
... ... ... ... ...
RS
...
1.65
...
1.0 1.65
... ...
1.15
5.5 0.89 0.4 0.72 0.68
..
..
.. ..
...
1.5 2.8
... ...
1
.o
0.65 1.50 0.90
...
Rsd
... ... ... ... , . .
8.4 4.6
...
5.9 4 0
...
0 89 0.49 1.4 0.79 0.40
0.71 0 45 1.0 0.55 0.41 1.44 1 24
o:i6
0 : 074
1.32 0.66 0.78
a a b b b
3 a-Hydroxy-5p-pregnan-20-one 38-Hydroxy-5a-pregnan-20-one 38- Hydroxy-5-pregnen-ZO-one 3p-Hydroxy-5.16-pregnadien-20-one 5a-Pregnane-3.11,20-trione 5p-Pregnane-3,I 1,20-trione 4-Pregnene-3,11,20-trione
, .
.. ..
... ... ...
...
1.3 1.3
, . .
3.5 3.5 2.7 I , 65 2.7
...
...
b b e e d d a a h
2.7 2 7 l,95 1.65 1.65 1.65 1 25 1.25 1.00
... ... , . . ... ...
3a-Hydroxy-5p-pregnane-l1,20-dione 38-Hydroxy-5a-pregnane-1 1,2O-dione 36-Hydroxy-5p-pregnane-l l ,20-dione 11a-Hydroxy-5a-pregnane-3,2O-dione 11a-Hydroxy-5p-pregnane-3,20-dione
a a a a a
0.79 0.64 0.79 0 64 0.79
1.6 1.6 1.6 1.6 1.6
llp-Hydroxy-5p-pregnane-3.20-dione Gp-Hydroxy-4-pregnene-320-dione 11u-Hydroxy-4-pregnene-3,ZO-dione 11a-Hydroxy-4,16-pregnadiene-3,20-dione 11a-Acetoxy-4-pregnene-3,20-dione
a b b b b
1.0 0.63 0.4 0.4 1 25
1.5 1.0 1 .o
1lj3-Hydroxy-4-pregnene-3,ZO-dione
b b
0.60 0.60
1.8 1.8
.. ..
...
... ... ...
...
... ... ...
... ...
1.2
0.49
... ... ...
... , . .
... ... ... ...
... , . .
...
0.43 0 25 0 53 0 33 0 22
0.19 0.06 0 26 0 12
0 90 0.82
..
Or87 0.82
0.52 0.34
.. .. ..
... ... ...
0.64 0.520
0.15 0.07 0.13
..
... 1.6
1.86 0.40 0.35
... ...
..
...
1.42 1.32 0.68 0.32
... ... ...
...
...
...
...
... ...
... ... ... ...
1.9
, . .
2.1
... ... ... 0.4 0.148 0.14
...
..
0.83 0.83 0.76 0.55 0 68
... .. ... ... ...
... ...
... ...
...
...
, . .
...
...
... ...
...
... ... ...
6.0
...
, . .
2.1 1.9
...
...
0.82 0.748 0.62 0.31 0.56
...
8.4
.. ..
0.33 0.168
4.0
... ...
0.99 0.52 0 28 0.52 0.43
1.66 1.558
... ... ... ...
0.99 0.74 0.63 0.80 0.78
;:A58
0.21
...
..
0.85 0.22 0.043 0.13 0.11
..
0.16 0.09 0.20 0.28
..
0.83
...
0.13 0.065 0.13 0.21 0.18
We
...
...
...
...
Bush B-5,
0 : 78 0.78 0.78 0.65
...
0’89
...
1.1 0.61 1.1 1.7 1.39
1.7 3.6
..
Rie
~
Bush B-3. Rle
0.74 0 69
0 65 1.0
... ...
~
Bush B-1,
0 22 0.11 0 83 0 85 0 99
0.57 0.64 1.3
...
~
... ... ...
...
2:9 5.3 8.7
... ... ... ...
..
...
1 51
1.80 1.800 0.38 0.82 1 .05
...
0 76 0.5 0.27 0.5 0.38
4 8
.. ..
...
... ... ... ... 1.44 1.23 1.61
1.15 0.740
...
HI e
E:;
...
...
!
Hs b
2.0
...
b d h d b b b
... , . .
...
0.74 0.08 0.47 0.19 0.11 0.04
... . .
0.95
... , . .
,..
0.42
0.89 0.89 0.82
0.14 0.11 0.16 0.08 0.12
..
0.21 0.12
0.68 0.76 0.72 0 72 0.64 0.61 0.42 0.25
o:i3 0.21
...
...
... ... ...
...
...
... ...
0.74 0 52 0.43 0.21 0.05
.. .. ..
.. .. ..
..
.. ..
.. .. 0.67 0.47 0.58 0.74 0.67
.. ..
..
0176 0.66 0.39
..
..
0 : 96 0.78 O.9G
..
0.90 0.96 0.93 0.89 0.78 0.78 0 78 0.78 0.65
.. .. ..
.. .. ..
.. ..
0.43 0.34 0.43 0.34 0.43
.. .. .. ..
2.1
...
3.3 1 6 1.0 1.0 5.3
1.12 1.0 1.04
0.69 0.47 0.47
...
0.65 0.34 0.19 0.19 0.71
.. ..
...
0.20 0.097 0.034 0.04 0.25
2.1 2.1
1.42 1.32
0.11 0.078
0.71 0.69
0.43 0.34
.. ..
...
...
, . .
..
,.
..
preferred methods of detection are indicated. 2,4-Dinitrophenylhydraainereagent Ultraviolet absorption and 2,4-dlnitrophenylhydraeine reagent Ultraviolet absorption Phosphomolybdic acid reagent 2.4-Dinitrophenylhydrazineand phosphomolybdic acid reagents and Tollens reagents Ultraviolet absorption, 2,4-dinitrophenylhydrazine, 2,4-Dinitrophenylhydrazineand Tollens reagents Ultraviolet absorption and Tollens reagents I. Tollens reagent b Relative mobility t o 4-pregnene-3,11,20-trione. c Relative mobility t o 11a-hydroxy-4-pregnene-3,ZO-dione. d Relative mobility t o 17a,21-dihydroxy-4-pregnene-3,1 1,eO-trione. e Relative mobility t o solvent front. I Dots indicates t h a t mobility f o r R/ values has not been determined because of low polarity for the system, polarity too high for the system, or extreme streaking. 8 Streaked on this system. a
Only a. b. c. d. e. f. g. h.
3.6
2.0 1 35
CISsteroids 4-Estrene-3,17-dione Estrone 17p-Hydroxy-4-estren-3-one Estradiol 11a-Hydroxy-4-estrene-3,17-dione 10~,17p-Dihydroxy-4-estren-3-one 11a,178-Dihydroxy-4-estren-3-one
14a-Hydroxy-4-pregnene-3,20-dione
... ...
...
14a,17p-Dihydroxy-4-androsten-3-one
4,9 (1l)-Pregnadiene-3.20-dione 4,16-Pregnadiene-3,20-dione
...
5.3
...
b b b b b
1,l-Pregnadiene-3,2O-dione 4,6-Pregnadiene-3,20-dione
RsC
1.50 1.0
b b d b b
D. C ~ steroids I 5a-Pregnane-3 .ZO-dione 5p-Preenane-3,20-dione 4-Pregnene-3,ZO-dione
Del-elopnient Systems Bush FBF, A
K-I,
0.89 0.60 3.5 4.25 5.85
17a-Rlethyl-I 78-hydroxy-4-androsten-3-one 17a-Ethinyl-178-hydroxy-1-androsten-3-one 5-Androstene-38,17p-d1ol 17P-Hydroxy-4-androstene-3,l I-dione l?n-hfethyl-17p-hydroxy-4-androstene-3,1 I-dione
C.
~
1857
V O L U M E 2 8 , NO. 1 2 , D E C E M B E R 1 9 5 6 ~
Table 111.
PT. RsC
Detrctiona
CAl,
15a-Hydroxy-4-pregnene-3.20-dione 15&Hydroxy-4-pregnene-3,20-dione 1Ba-Hydroxy-4-pregnene-3,2O-dione 17a-Hydroxy-4-preenene-3,2O-dione Zl-Hydroxy-4-preenene-3,ZO-dinne
h h b h
0.37 0.4 0.4 0.78 0.86
1.0 1.1 0.8 1.8
Methyl-3,l l-diketo-4,17(2O)-(cis)-pregnadien-21-oate la. 1 1a-Dihvdroxv-5E-nrrnnan-2O-nne
b a a b b
1.5 0.4 0.24
...
Compound
3a,17a-Dihydroxy-50-pregnane-l1,ZO-dione 6&11a-Dihydroxy-4-pregnene-320-dione 68,17a-Dihydroxy-4-pregnene-3,20-dione 11a,17a-Dihydroxy-4-pregnene-3,2O-dione 1la, 17a-Dihydroxy-4-pregnene-3,20-dione
E.
Steroid hlobilities (Continued)
C2121-Iiydroxpsteroids 2 l-Hpdroxy-4-pregnene-3,20-dione 2 l-Arrtoxv-4-nreenene-3.2O-dione
f
h b b a a b b b b
f f f
f f
68.2 l-Dihydroxy-4-pregnene-3.20-dione 1 1a.2 l-Dihydroxy-4-pregnene-3,20-dione 11/3,21-Dihydroxy-4-pregnene-3,20-dione l4a.2 l-Dihydroxy-4-pregnene-3,2O-dione 15a.21-Dihydroxy-4-pregnene-3,2O-dione 17a.21-Dihydroxy-4-pregnene-3,20-dione 17a-Hydroxymethyl-1 7aa-hydroxy-D-homo-4-androstene3.17-dione (D-homo compound S ) 21-Acetoxy-1 l@-hydroxy-4-pregnene-3,2O-dione ?l-Acetoxy-l5a-hydroxy-4-pre~nene-3,2O-dione 2 l-.lcetouy-l7a-hydroxy-4-pregnene-3,20-dione 1 i a 21-Ditiydroxy-SP-pregnane-8,11 ,ZO-trione 17a 21 -Dihvdroxv-4-nrennene-3.11.20-trione
RSb
6:8
... ...
..
...
0: 29
...
0:i4 0.4
0.91 1.5 1.0
.. ..
.. ..
...
0 : 074
...
0.14 0.38
0.86 1.4 1.15 0.28 0 47
...
..
f
0:22 n. 17
0.63 0 42
n:is
0:i2
0:4
0.71 2.6 0.92 1 8
f f f b f f f
if"
;
0:49
0.23
..
0.21 0.16
n:i
0.22
h f f
... , . .
1
58
O.9lQ
..
f
h
21-hcetoxy-3a. 17a-dihydros i 21-.lcetoxy-R~,l7rr-dihvdrox f 21-Acetoxy-l1p, 17a-dihydro 21-Acetosy-1 lp. 17a-rlihydroxy-l,4-pregnadiene-3,20-dione h 11/3,17a.?"a.2 l-Tetrahydroxy-i-preenen-~-one 1l p , 17a.208.21-Tetrahydroxy-4-pr~pnen-7-one 21-.4cetory-l1~,17a,20a-trihydroxy-i-pregnen-8-one 21-,~cetoxy-4/3-bromo-17a-hydrnxy-5p-pregnane-3,11.20trione 2-Alethyl-1 Ip,l7a,21-trihydroxy-4-pregnene-3,2O-dione
..
h b h
..
s
0.4
21-.~cetoxy-9or-fluoro-i 1~,17a-dihydrox~--4-pregnene-3.20f dione 2-1\Iethyl-?l-acetosy-l l g , l 7 a - d i h y d r o x ~ - i - p r e g n e n e - 3 , 2 ~ ~ f dione 17a,21-Dih~-droxv-58-prr~nane-7,11,20-trione-3.20a ethylene glscol diketal 17a,~1-Diliydroxy-5-pre~nene-3,11.20-trione-3,20a ethvlrne glycol diketal 1la.17u,?l-Trihydrosy-6-pregnene-3,2~-dione-~,20rrhylene glvcol diketal a 2-.\Irthyl-9a-flnorn-l1~.!7a.21-trihydroxy-~-pregnenef 3.20-dione 2-hlethyl-21-acetoxy-9a-fluoro-llii, 17a-dihydroxy-4f pregnene-3,20-dione
.. ..
..
... . . ... ...
.. 0.5
..
0.36 0.28
..
...
0.017 0 030 0 024 0 IC, 0.23
. .
0.57
0 74 0 63 0.47
..
0.57 1.8 1.0
0 58
...
...
1. o 2 9
0.2: 0.04 0.19 0.09 0.29
0'89 0,067 0 18 0.05 0 21 0 13 0 39
. .
4.0
1 . .i
i'o
1.9 0.61 1 7
...
...
...
... ..
0':
n:o;
0.304
... ... ... ... ... ...
0.23 0.53 0 50
0.28 n 16 n 49 0.36 0 I2 0 36
0 19 0 13 n 39 0 23 n ,080 0.35
...
G 4
0.38 2 1 0.73 1.7
0 3.5 136 0.79 I 3
, . .
1 . .5 1.0 0 on 9.50 6.oe
0.11 0.08.5
0 11 0.0.5i
0 : 640
.o
...
0 03
4.4g 0 23 0.12 0.44 n 27 I d 1 1 2 90 1.30
0.398
0,568
i o 1 n.033
0'01 0 029 o 018 0 31 0 21 0 390 0 250
..
1
o'ii 0 08 0 0 158
0 09 0.12 1. 0
..,
13.9e 0.86
...
1.40
:
...
0 6.55
...
1 42 0.09
0.78 0.81
0 13 0.20 0.15 0.52 0.52
...
0 78
0.41 0.29 0.31
...
0 28
n.~.3
... , . .
...
...
... ...
0 ' 78
n‘17
0 47 0 05 n 32 0 04 0.19 0.09 0 23
0.19
0.81
0.52 0.89 0.89
...
...
0 21 0.10 0 33 0 21 0.06 0.33
3: 1.I 4.6 3 5 1.36 3.5
...
0.40 0 49 n -13
0.43 0.75
4.1
...
Bush B-3. R/e
...
O'k9 Ir,
1:j
Bush B-1. E.;. e
0:10 .. .. ..
Rllsh B-5,
E/
e
..
..
0 : 87 0.87 0.87
..
..
..
0147 0.87 0.38 0.75 0 61 0.83 ,.
.. 0'92
0:43
..
..
0 75 0.61 0.87 0.77
..
.. .. ..
..
0.53
0.87
0.30 0.71 0.34 0.73
0'38 0 10 0.43
0 21 0.09
.. ..
. . ...
o'fiba 0.44
0 :2 8 0 0 15
... . . ... . .
o
0.12
0.w
.. ..
0.12 0.37 0.27 0.87 0.87 0 9 0.828
...
.., ...
... ..,
..
...
... ...
. . . .
n
4n nz4
0 013
n o
n4 n2.i 0 28 0 21 n 340 0.270
0'09 0.07
... ...
.. ..
, . .
0.09
...
...
0.71 0.10
..
..
0.39
0 87
..
.. ..
0.75 0.61 0.57
0:95
n 220
0.09 0 12 0.61
..
0:59
...
0.23
...
0 278
..
0.9
6.18
0 64
0.92
0.51
0.20
0.96
...
1.7
1.26
... ...
0.60
0.37
..
...
1.4
1.0
0.47
0.23
0.91
1.7
0.66
0.5
... ...
0.31
0.13
0.61
0.04
...
0.057
..
0.61
0.418
0.15
0.95
0.44
...
4.49
0.49
0.568
preferred methods of detection are indicated. ?.4-Dinitrolihenylhydrazine reagent Utraviolet absorption and 2.4-dinitrophenylhydrazine reagent L-ltraviolet absorption Phosphomolybdic acid reagent P. 2.4-Dinitrophenylhydrazineand phosphomolybdic acid reaeente f . Ultraviolet absorntion. ?,4-dinitroplienglhydrazine, and Tollens reagents p. 2.4-Dinitrophenylhydrazine and Toilens reagents h . 1-lrraviolet absorution and Tollens reanents i. Tollens reagent b Relative mobility to 4-pregnene-3,11.20-trione. C Relative mobility to 1 la-hydroxy-4-pregnene-3,20-dionc. d Rela t i r e mobility t o 17a,2 l-dihydroxy-4-pregnene-3,11.20-t rione. Relative mobilitv t o solrent front. f Dots indicates t h a t mohility for R I values has not been determined because of low polarity' for the system, polarity ton high for t h e system. or extreme streaking. 0 Streaked on this system. a
Only a. h. e. d.
... 0 :i7
5.8
0.82 1.0 0.74 1.31 1. 3
0.43 0.29 0.29 4,8
.
f
f
...
0 77 1.2 0.92 2.53 4 0
3.0 2.3 2.3
ii:o
.. ,
RSd
0.8
i:i5
0:i5
PTF.
Development Systems Bush FRF. -1, IC-1, R s h El. e RSC
1858
ANALYTICAL CHEMISTRY
photometer at 22.2 and 242 nip. The calculation ( 1 0 ) for 11 a-hydroxy-4-pregnene-3,20-dione is carried out as described in the above formula. Contact with rubber must be avoided a t all stages after the paper strip has been developed. The 4-pregnene-3,20-dione is determined in a similar fashion. As the amount of 4-pregnene-3,20-dione present as a contaminant in the 1la-hydroxy-4-pregnene-3,20-dioneobtained by microbiological conversion is in the vicinity of 1%, it is necessary to streak 10 mg. of sample across a 3-inch space of a chromatogram prepared for the Carbitol-methylcyclohexane system. Bfter overnight development, the papergram is dried and the area is cut out and eluted, filtered, and read. I n one study for evaluation of the accuracy of this method, four samples in the range of 90 to 95% lla-hydroxy-4-pregnene3,20-dione were assayed with six replicas of each analysis. The recovery averaged 100 zt 0.5% with no significant difference a t the level of confidence of 19 out of 20 samples. For 4-pregnene3,20-dione at the same level, two samples with six replicates each gave 99.7 zt 1.1%. These results are comparable to those obtained by infrared analyses. When pure 4-pregnene-3,20-dione was applied to duplicate the amount present in a n 1la-hydroxy-4-pregnene-3,20-dione sample containing 1 to 2% of 4-pregnene-3,20-dione as an impurity, the average recovery was 96.6 zk 275, in two samples with six replicates each. It would be very difficult to work out an infrared analyeis for 4pregnene-3,aO-dione present in crystalline 1lahydroxy-4pregnene-3,20-dione with this accuracy.
Blend 1
2 3
Table IV. Analyses of Crystalline lla-Hydroxy-4-pregnene-3,ZO-dione 11a-Hydroxy4-pregnene4-PregneneOther 3,20-dione 3,20-dione (Visual) 95.8, p 9 . 2 2.0,2.1 1.0 (92.3) (1.73)a (98.7) b (2.29)b 100.6, 99.0 1.55, 1.34 1 4 99.2, 99.2 1.58, 1.62 1 8
Percentage present in sample if lower extreme of 1.7 for blank ratio is used in formula for calculation. b Percentage present if higher extreme of 2.9 for blank ratio is used.
Table IV shows typical results on three different blends of crystalline 1la-hydroxy-4-pregnene-3,2O-dione. It is assumed ~ Z for extraneous material elutable that the ratio of D Z N / D Zmp from the chromatogram by absolute methanol (blank correction) is a constant. The range of values determined on 66 samples was 1.7 to 2.9 with an average of 2.16. This is introducing an error into the determination, but i t seems more logical to take an average value determined from a large number of samples of one lot of filter paper than to take a single sample from a fragment of the same lot (which is done with a single blank determination from the same strip), The need for a control is also eliminated because K for any given steroid is a constant.
Furthermore, the method is very flexible for any quantity of steroid from 50 y to 10 or more mg. It can be applied to any steroid having ultraviolet absorption (xith appropriate choice of m-ave lengths for reading the absorbance).
ACKNOWLEDGMEhT
This work could not have been completed n-ithout the technical assistance given by Jennie Rlejeur Noteboom, Henrietta Triemstra, and Hester Roltersom. The author also wishes to acknowledge the continued interest and help of D. H. Peterson, &I.E. Speeter, and R. H . Levin. The statistical study waa generously done by Burton D. Seely. The thoughtful suggestions and criticisms of S. H. Eppstein and J. L. Johnson are greatly appreciated. LITERATURE CITED
(1) Axelrod, L. R., J. Biol. Chem. 201, 59 (1953). (2) Ibid., 205, 173 (1953). (3) Axelrod, L. R., Recent Progr. Hormone Research 9, 69 (1954). (4) Axelrod, L. R., Brroyave, G., J . B i d . Chem. 208, 579 (1954). (5) Bush, I. E., Bwchem. J . 50, 370 (1952). (6) Bush, I. E., Recent Progr. Hormone Research 9, 321 (1954). (7) Drake, iY.h.,Haines, W. J., Knauff, R. E., Nielson, E. D., AN.4L. CHEhf. 28, 0000 (1956). (8) Eberlein, W.R., Bongiovanni, A. hl., Arch. Biochem. and Biophys. 59, 90 (1955). (9) Huesghem, C., .Yature 171, 42 (1953). (10) Johnson, J. L., Struck, 1%'. d.,Scott, E. J., Stafford, J. E., ANAL. CHEM.25, 1490 (1953). (11) Kochakian, C. D., Stidworthy, G., J . Bid. Chem. 199, 607 (1952). (12) Kritchevsky, D., Kirk, RI. R., Arch. Biochem. and Biophys. 35, 346 (1952). (13) Kritchevsky, D., Kirk, 11. R., J . Anz. Chem. SOC. 74, 4484 (1952). (14) hZcDonough, S , Nature 173, 645 (1954). (15) Mattox, V. R., Mason, H. L., Albert, A,, J . B i d . Chem. 218, 359 (1956). (16) Murray, H. C., Peterson, D. H. (to Upjohn Co.), U. S. Patent 2,602,769 (1952). (17) Seher. R.. Wettstein, A., H e h . Chim. Acta 35, 276 (1952). (18) Pechet, hI. hI., J . Clin. Endocrinol. and Metabolism 13, 1542 (1953). (19) Pechet, &I. XI., Science 121, 39 (195f). (20) Peterson, D. H., Eppstein, S. H., Neister, P. D., Murray, H. C.. Leigh, H. >I., Weintraub, A., Reineke, L. AI., J . Am. Chem. Soc. 74, 451 (1953). (21) Rubin, B. L., Dorfman, R. I., Pincus, G., J . Biol. Chevz. 203, 629 (1953). (22) Sakal, E. H., RIerrill, E. J., Science 117, 451 (1953). (23) Savard, K., J . B i d . Chem. 202, 457 (1953). (24) Savard, K., Rscent Progr. Hormone Research 9, 186 (1954). (25) Schindler, O., Reichstein, T., Helv. Chim. Acta 34, 108 (1951). (26) Shull, G . h l . , Bardinas, J. L., Subel, R . C., Arch. Biochem. and B i o p h y s . 37, 186 (1952). (27) Zaffaroni. A , Recent Progr. Hormone Reseaich 8, 50 (1953). (28) Zaffaroni, d.,Burton, R. B., J . Bid. Chem. 193, 749 (1951). (29) Zaffaroni, rl., Burton, R. B., Keutman, E. H., Ibid., 177, 109 (19491. (30) Ibid., 188, 763 (1961). (31) Zaffaroni, 9., Burton, R . B., Keutman, E. H., Science 111, 6 (1950). RECEIVED for review August 1, 3966.
rlccepted October 4, 195F
