Trimethyl Silyl Ether-Enol-Trimethyl Silyl Ether- A New Type of Derivative for the Gas Phase Study of Hormonal Steroids E. M . Chambaz, Genevieve Defaye, and Christian Madani Biochimie Endocrinienne, C.E.R.M.O., Universite Scientifique et Medicale, Grenoble, Domaine Universitaire, Saint-Martin-d’Heres (381, France
Conditions for a base-catalyzed silylation procedure using nucleophilic agents are described. This type of catalysis leads to the preferential formation of enol TMS from steroid keto groups without affecting the silylation rate of hindered hydroxy groups. Considerable differences are seen in the reaction rate with different silylating agents and several nucleophilic catalysts are effective. The most easily derivatized structures are the keto1 and the dihydroxy acetone side chains of the corticosteroid metabolites. Mass spectrometry and NMR studies demonstrated that the side chain remains in place with a C-20-C-21 unsaturation. Applicability of this new type of derivative for the gas phase study of hormonal steroids has been investigated and appears of great interest, specially for the study of adrenal steroid metabolites in humans.
Trimethyl silyl ethers (TMS) are very widely used in the gas phase analysis of steroids since their introduction by Luukkainen et al. ( I ) . Several reports have established the conditions leading to the silylation of hindered hydroxy groups (2, 3 ) . More recent works have developed persilylation procedures for gas-liquid chromatography (4, 5). Strong silylation conditions can be obtained by increasing the amount of “catalyst” (usually trimethyl chlorosilane) and/or the reaction temperature (2-4). Aringer et al. have recently reported more potent catalytic activity exhibited by trimethyl bromosilane (5). Using this type of acid catalysis, the formation of enol-TMS from the keto groups of the steroids was shown to occur, although a t a different rate according to their environment in the molecule (5, 6). These enol-TMS are generally not formed in 100% yield and may, therefore, appear mostly as undesirable products for analytical purposes, so the formation of a keto derivative (e.g., methoxime) was generally suggested for quantitative work (3-6). In addition, these strong silylating conditions may lead to additional reaction products via an oxisilylation process (6). We have investigated the effect of a base-catalyzed silylation procedure, and some nucleophilic agents were found able to promote the enolization of steroid keto groups. A new type of derivative (TMS-enol-TMS) was proposed for the gas phase study of saturated corticosteroid metabolites possessing the dihydroxy acetone side chain (7, 8). This paper reports further studies on the structure and formation of Luukkainen, W. J. A. Van DenHeuvel, E. 0. A. Haati, and E. C. Homing, Biochim. Biophys. Acta, 52, 599 (1961). E. M . Chambaz. and E. C. Homing, Anal. Lett.. 1, 201 (1967). E. M . Chambaz and E . C. Homing, Anal. Biochern., 30, 7 (1969). (4) N. Sakauchi and E . C. Horning, Ana/. Lett., 4, 41 (1971). (5) L. Aringer. P. Eneroth, and J. A . Gustafsson, Steroids, 17, 377 (1971). (6) E. M . Chambaz. G. M . Maume, B. Maume, and E . C. Horning, Anal. Lett., I , 749 (1968). (7) E. M . Chambaz and C. Madani, Excerpta Med. Int. Congr. Ser.. 210,97 (1970). (8) E. M . Chambaz, C. Madani, and A. Ros, J. Steroid Biochern., 3, 741 T.
(1972).
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this derivative and examines its possible applicability to other steroidal structure of hormonal interest.
EXPERIMENTAL Model steroid compounds were from a commercial source (Mann Research Laboratory New York, N.Y.). Trivial names of steroids and abbreviations used in the text are as follows: Androsterone (An): 3a-hydroxy-5a-androstan - 17-one; Cortisone ( E ) : 17a,21-dihydroxy-pregn-4-en-3,11,20-trione; Cortisol (F): 11~,17a,21-trihydroxy-pregn-4-en-3,20-dione; Deoxycortisol (S): 17a,21-dihydroxy-pregn-4-en-3,20-dione; Deoxycorticosterone (DOC): 21-hydroxy-pregn-4-en-3,20-dione; Corticosterone (B): Ilp,21-dihydroxy-pregn-4-en-3,2O-dione; Tetrahydrocortisone (THE): 3a,l7a,21-trihydroxy-5p-pregnan-l1,20dione; Tetrahydrocortisol (THF): 3a,llp,17a,21-tetrahydroxy5p-pregnan-ZO-oneand its 501 isomer (allo-THF); Tetrahydrodeoxycortisol (THS): 3a,l7a,21-trihydroxy-5P-pregnan-20-one; Tetrahvdrodeoxvcorticosterone ( T H DOC): 3a,Zl-dihydroxy-5@pregnan-20-one; Tetrahydrocorticosterone (THB): 3a,ll&21trihydroxy-5p - pregnan-20-one; Tetrahydrodehydrocorticosterone (THAI: 3a,Zl-dihydroxy-5p-pregnan- 11,20-dione; Cortol: 56pregnane-3a,llp,l7a,ZOa,21 - pentol; Pregnanolone: 3a-hydroxy5p-pregnan-20-one; 17-OH-pregnanolone: 3a,l7a-dihydroxy - 5ppregnan-20-one; Cholesterol butyrate (C. But.): 3p-hydroxy-cholest-5-en-3-n-butyrate. The silylating agents: BSA ( N ,0-bis(trimethylsilyl)acetamide), BSTFA ( N ,0-bis(trimethylsilyl)trifluoroacetamide),TSIM (trimethyl silyl imidazole), HMDS (hexamethyl disilazane), TMCS (trimethyl chlorosilane) were from Pierce Chemical Co., Rockford, Ill. All other chemicals, A grade, were purchased from Merck, Prolabo, or Fluka. Gas chromatographic analyses were performed on Carlo Erba Instruments (GI and GV models), with 6- or 12-foot silanized glass columns filled with 100-120 mesh gas chrom P coated with 1% (w/w) stationary phase (OV-1 or OV-101 and OV-17), according t o Horning et al. (9). The studies of the reaction rates were performed by isothermal GLC analysis; retention parameters were determined by temperature programming and expressed as methylene units (MU) as previously described (9). All reactions were carried out in tightly capped small test tubes. Noncatalyzed silylation was carried out on 50 pg of steroid with 200 pl of BSA or BSTFA overnight at room temperature (3). For the base catalyzed reaction, 5 to 40 mg of potassium (or sodium) acetate in methanolic solution were evaporated to dryness under a nitrogen stream in the reaction tube; 50-100 pg of the steroid were then added; after complete evaporation of the solvent, 200 pl of silylating agent (BSTFA, BSA, or TSIM) were added. Two to 4 pl of the mixture were either directly injected into the gas chromatograph (or the gas chromatograph-mass spectrometer) or evaporated under a nitrogen stream and taken up in hexane (for direct injection mass spectrometry), carbon tetrachloride (for nuclear magnetic resonance study) or HMDS (for gas chromatography or storage). For the quantitative study of the reactions, cholesterol butyrate (50 to 100 pg) was added as an internal standard prior to the silylating agent. Mass spectra were obtained on an LKB 9000 gas chromatograph-mass spectrometer. The separator was kept a t 250 “C. In all cases, the spectra were recorded a t 70-eV ionization energy as well as 20-eV energy, in order to obtain a better definition in the high masses range. Some of the spectra were recorded on an AEI (9) E. C. Horning, M . G . Homing, N . Ikekawa, E. M . Chambaz, P. I. Jaakonmaki, and C. J . W. Brooks, J. Gas Chrornatogr., 5, 283 (1967).
I 1 % ov-101 12 f t
z-22 190 x 1.2'C/min THE TMS m0l-TM.S I BSTFA)
:-24
:-28
32
C-26
THE
1
MU: 30.98
J
-
1 Y. ov-101 12 f t
190
x
12'C/min
i
allo-THF TMS end-TMd (BSTFA)
c-22
I
I
Figure 1. Gas chromatographic analyses of the derivatives obtained from THE (upper trace) and a-THF (lower) with BSTFA in the presence of potassium acetate
Straight chain hydrocarbons (C-22 to C-32) were included in these temperature-programmed analyses for methylene unit determination. The same amounts of steroid and cholesterol butyrate (CBu) were used for the quantitative study of the derivative formation
MS-9 instrument, with direct injection, a t 70 eV, the source being kept between 150-250 "C. Nuclear magnetic resonance (NMR) spectra were recorded on a Varian A-60 instrument, in carbon tetrachloride, with tetramethyl silane as internal standard.
RESULTS Saturated Pregnane Steroid Metabolites Possessing a 20-Keto Group. 20-21 Keto1 Side Chain and 17a,21Dihydroxy-20-Keto Side Chain. The major metabolites of biological interest in these categories were taken as model compounds: THA, THB, THS, T H E , THF, a-THF. While the ketolic side chain metabolites (e.g. THA, TH-DOC, THB) yield convenient TMS derivatives under
noncatalyzed conditions, the dihydroxy acetone side chain structure under the same conditions does not give a thermally stable product (7-10). By contrast, when the silylation reaction (BSTFA) was carried out in the presence of a nucleophilic agent such as potassium (or sodium) acetate (overnight a t room temperature), a single product was obtained with excellent GLC behavior. Figure 1 shows the GLC analysis of the derivatives obtained for T H E and a-THF, two of the major urinary corticosteroid metabolites. (10) E. Bailey in "Gas Phase Chromatography of Steroids," K . B. EikNes and E. C. Horning, E d . , Springer Verlag, New York, N.Y., 1968. p 316.
ANALYTICAL CHEMISTRY, VOL. 45, NO. 7 , JUNE 1973
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T H E . BSTFA 120
o/~
chol. bu.
3s /
100
--
50 TMS enol TMS f o r mat ion
Figure 2.
Rate of the derivative formation for THE with various amounts of potassium acetate
Yields were calculated (peak areas) with regard to the internal standard (cholesterol butyrate)
Table I. Retention Times (Expressed as Methylene Units: M U ) of the Main Human Urinary Saturated Corticosteroid Metabolites MU values
Tetrahydrodehydrocorticosterone ( T H A ) Tetrahydrocorticosterone (THB)
Tetrahydrodeoxycortisol (THS) Tetrahydrocortisone ( T H E ) Tetrahydrocortisol (THF) Allo-THF (a-THF)
Cortol
1% OV-1
1% OV-17
31.29
32.36
31.56 29.81 30.98
32.78
31.24 31.54 32.66
29.92 31.58 32.02 32.15 33.91
Table I gives the retention parameters obtained for the different model compounds after silylation under acetate catalysis. The quantitative aspect of the reaction was studied by reference to a stable internal standard. Cholesterol butyrate was used and a reproducible response coefficient (100 to 120%), was regularly found for the compounds investigated. Cortol was treated in the same manner to see if the base catalyzed silylation was effective on the hindered 116- and 17~-tert-hydroxylgroups. The retention data (Table I) as well as the mass spectrometric analysis, indicated a cortol tri-TMS. Therefore, the process promoted by the nucleophilic agent is not equivalent to the usual acid catalysis (2, 3). The influence of the amount of acetate on the reaction rate was investigated with T H E (50 pg) and BSTFA (200 pl) as the silylating agent. Figure 2 shows that no clear relationship was evident between the amount of potassium acetate (10 to 40 mg) present. However, this might be caused by the heterogeneous reaction mixture. For further studies, 10 or 20 mg were usually used, since the conversion was not complete after 22 hours if only 5 mg of acetate was used. 1092
ANALYTICAL CHEMISTRY, VOL. 45, NO. 7, J U N E 1973
Figure 3 shows the reaction rate for T H E a t different temperatures: 6 hours a t 50 "C was sufficient for a complete reaction, but the same yield was obtained after a 24-hour reaction under either condition. Figure 4 gives the rate of the derivative formation for THS, THE, and T H F a t 25 "C. This result suggested an influence on the base catalyzed process brought by the nature of the substituant a t the 11 position. However, the yield was maximum in either case after a 24-hour reaction. Figure 5 shows the mass spectra obtained for the T H S and T H F derivatives. In all cases, a good molecular ion indicated a tetra-TMS derivative. In addition to the loss of a trimethylsilanol (M - go), the common main feature in these spectra is a prominent ion a t m / e 331. This can be attributed to a cleavage of the D ring between C-13C-17 and C-144-15, yielding a fragment bearing 3-TMS groups, as indicated on the figure. This was confirmed after derivatization using a deuterated silylating agent (Dls-BSA). The deuterated T H S derivative showed a shift of 36 amu for its molecular ion (4-TMS), the ion a t m / e 331 being shifted to 358 (+ 27 amu) indicating 3-TMS groups (Figure 5 bottom). These features can be understood on the basis of the proposed structures (Figure 5) and led to name this type of derivative TMS-enol-TMS. Figure 6 gives the mass spectrum for the derivative obtained from tetrahydrocorticosterone (THB). A 20-enolTMS, 21-TMS structure can explain the molecular ion (tri-TMS) and the prominent ion at m / e 203 by cleavage of the D ring. Steroid Metabolites with a 20-Keto-21-Methyl Side C h i n . Pregnanolone was used as a model compound. BSTFA as well as BSA in the presence of acetate never led to a single reaction product. A 72-hour reaction a t room temperature yielded about 40% of the di-TMS derivative ( M + 462) attributed to the 3-TMS-20 enol TMS structure according to the mass spectra, the other product being the 3-TMS derivative. Substitution a t (2-21, as given by the 21-OH groups in hormonal steroids appears necessary for the quantitative formation of the enol TMS from the 20-ketone.
+
oh
chol. bu.
1GO / / /
T H E - BSTFA Kac.10 mg
TMS enol TMS formohon
I
1
2
3
4
5
6 7 HOURS
8
9
// //
24
Figure 3. Rate of derhative formation for T H E (BSTFA, potassium acetate) at different temperatures
BSTFA K a c e t a t e 2 0 m g
25
chol. bu.
I
1
./
'c
/.-AC
-THS
-
/
/
I
1
I
I
HOURS
3
I
4
Figure 4. Rate of derivative formation from THS, THE, and T H F (BSTFA, potassium acetate)
Corticosteroids with a 3-Keto-4-ene Structure. Deoxycorticosterone (DOC), corticosterone (compound B), deoxycortisol (compound s), cortisone (E), and cortisol (F) were used as model compounds. The formation of two enol-TMS isomeric products was expected from the 3-keto group, by analogy to the results obtained with acid catalysis (5, 6). Figure 7 A shows that it was indeed the case: cortisone treated with BSTFA in the presence of K acetate yielded three products: e l and e2 represented tetraTMS derivatives and were believed to be two isomeric 3enol-TMS, possibly with a 2,4 and a 3,5 diene structure, respectively. When the reaction mixture was evaporated to dryness and taken up in c c l 4 or hexane (Figure 7 B ) , a major single product was obtained, corresponding to a
TMS-enol-TMS with a free 3-keto group. This was attributed to the rapid hydrolysis of the T M S on the enol a t the 3 position during the evaporation-redissolution steps in the absence of silylating reagent. The TMS-enol-TMS obtained in this manner could be redissolved in HMDS; under these conditions, it was stable for a t least one week at room temperature. NMR study was carried out on the cortisone derivative after redissolution in CC14, Figure 8 gives the NMR spectrum obtained. The signals at 5.85 and 5.60 ppm assigned to the protons a t C-4 and C-21 are in agreement with the structure proposed for the dihydroxy acetone side chain TMS-enol-TMS derivative, with a C-20-C-21 double bond. A N A L Y T I C A L C H E M I S T R Y , VOL. 45, NO. 7 , J U N E 1973
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roo
TETRAHY DRO
1J71 Y
S. HC- O S i M e 3
70 e V
II
O S ; Me3
C-
t
-1 W
147
a
M 638
M- 90
169
548 1
-100
t IJl
z
w
f .- 50 w
2
c
147
5
W
a
Al
b
Il,llI 11 1
II
I,
rh
M-15
I
I
1 1
300
THS
32
l000% 16:
654
318
L
200
IO0
M
M-90 564
169
TMSenol TMS DI8
78
BSA
70 eV
50 209
a
261
4C
T C
3
I) 0
5 75
I
s
I
10
200
I
300
. "/e
I
1
,
600
65 6 1
I
700
Figure 5. Mass spectra of the derivatives obtained f r o m THS, and THF (BSTFA, potassium acetate) and the corresponding THS deuterated TMS-enol-TMS derivative (bottom: Dla-BSA)
TETRAHY DROCORTICOSTE RONE 100
C H OSi Me3
II
566
TMSenol TMS MeSiO
MW 566
3 u C
0 C
2 a
230
s 476 150
350
4 50
551
II 550
Figure 6. Mass spectra obtained for the derivative of tetrahydrocorticosterone
1% OV17 270 'C
K ac. + BSTFA 65mnt60"C
CORTISONE
C h o l Eu
Figure 7. Gas chromatographic analysis of the reaction mixture (A) of cortisone (BSTFA, potassium acetate). e l and e2 are tetra-TMS derivatives, believed to be the 2 isomers of the 3-enol-TMS structure. After evaporation and redissolution in H M D S (fl), a single major product is obtained with a free 3-keto group (T.e.T.1 Figure 9 gives the mass spectrum of the same cortisone derivative. The main features are in agreement with the proposed structure (see also Figure 4). Table I1 lists the retention parameters for some major unsaturated corticosteroids of biological interest, as TMS-enol-TMS derivatives (free 3 ketonic groups). Base Catalyzed Enolization of the Steroid 17-Keto Group. Figure 10 shows the evolution of the reaction mixture for androsterone treated, respectively, by BSTFA and BSA in the presence of K acetate. Considerable difference was seen between the effect of the two reagents. The proportion of the di-TMS derivative remained stable after 2 hours with BSTFA, and represented about 15% of the dervatives, while the 3-TMS 17-keto form of androsterone
was 85%. On the other hand, BSA promoted a progressive enolization a t the 17 position toward a 95-9770 yield after 24 hours. TSIM in the same conditions yielded only 3 to 5% of 17-enol-TMS, while HMDS did not give any detectable 17-enol-TMSproduct. Mechanism of the Reaction. In order to shed some .light on the reaction mechanism, the effectiveness of some possible nucleophilic agents was tested. Table I11 gives the yield obtained with T H E and BSTFA for the TMS-enolTMS derivative, expressed as the ratio to the internal standard. For these reactions, reagents in solid form were added (20 mg) to the silylating mixture, liquid reagents were used after mixing with BSTFA ( 1 : 2 or 1:l v/v). It was found that more convenient reagents than acetate ANALYTICAL C H E M I S T R Y , VOL. 45, NO. 7, J U N E 1973
1095
& 21
C K O Si M e3
:;I:; I
CC14
4
lr":
Mr
19
6 0 Mc
~
H4
I . . .
6
,.
. . I
5
.... I PPM(6) 4
.
I 3
.
.
. I
3
. - & . . . l . . . . I L . . I . . . . . - - - - ' . .
2
1
0
Figure 8. NMR spectrum of the derivative obtained from cortisone, after evaporation and redissolution in carbon tetrachloride 576
CORTISONE
T M S enol TMS
Di 150;
'i'
3?'
'7
600
Figure 9. Mass spectrum of the TMS-enol-TMS derivative of cortisone (evaporation and redissolution in hexane) were as efficient, particularly piperidine, which has some practical advantages (liquid form, volatile).
DISCUSSION The addition of a nucleophilic reagent either in solid or liquid form to a silylating agent promotes the formation of an enol-TMS derivative from the keto group of steroidal compounds. Although there are large differences in the behavior of the various silylating agents (e.g., BSA us. BSTFA), in either case the reaction does not appear as a usual catalyzed silylation since the hindered hydroxy groups (Ilp,17a-tert, e.g., in cortol) are not silylated under base catalysis. By contrast to the acidic catalysis (2, 3, 5, 6), these findings suggest a somewhat preferential effect on the enolization rather than on the silylation process itself. With regard to the yield of enol-TMS from the different keto groups investigated, this study suggests the following order: C-20 (with C-21 OH) > 3 CO-~?,r >> 17 > C-20 (C21-deoxy) >> 11. 1096
ANALYTiCAL CHEMISTRY, VOL. 45, NO. 7 , JUNE 1973
This is in contrast to the results of Aringer e t al. (5) using an HMDS-trimethyl bromosilane reagent mixture: their acid catalysis was effective in converting the 20-ketone in 21-deoxy compounds (e.g. pregnenolone) into an enol-TMS (98%). According to our results, the 20-21 keto1 and the dihydroxy acetone side chain were the structures the more easily converted to a complete TMS-enol-TMS derivative under base catalysis. In addition, we have no evidence of any enol-TMS formation from the 11-keto compounds studied. With regard to the enol-TMS formed, only the 3-keto4-ene structure yielded 2 detectable isomeric products. Another aspect is the stability of the 20-enol-TMS when transferred in solvents (hexane, CC14), while the 3-enolTMS are rapidly hydrolyzed with this treatment. This is in contrast to the relative stability of the %enol ester such as heptafluorobutyrate (11). This might be turned into a (11) D Exleyand J. Chamberlain, SteroJds, 10, 509 (1967)
Table II. Retention Times (Methylene Units: M U ) of Some Corticosteroid Hormones as TMS-enol-TMS (Free 3-Keto Group) MU values 1% o v - 1
11-Deoxycortisol (S) Cortisone ( E ) Cortisol ( F ) Deoxycorticosterone (DOC) Corticosterone ( B )
1% OV-17
31.78 32.76 33.14 30.98 32.79
33.69 31.06
BST FA
35.78 33.89 36.47
3T.17eL
t
Table Ill. Effectiveness of Some Reagents in Promoting the TMS-enol-TMS Derivative Formation from THE Catalyst
CH3COOK
CH3COONa HCOONa Na Citrate KzC03
Acetone Ethylacetate DMSO
Urea Glycine Pyridine Piperidine Formamide Phenylenediamine HCI Aniline Ethanolamine
1
2
hl-20
Yield, % a
110 110 27 102 0 0 0 14 79 29 0 110 110 15 0 0
aThe yield was evaluated with the use of an internal standard (cholesterol butyrate) and was expressed as the ratio to this standard (peak areas).
possibility to get a sole product after the reaction with A 4-3 keto compounds such as cortisone, after differential hydrolysis of the two 3-enol-TMS isomers, while the side chain TMS-enol-TMS remains stable. In any case, base catalyzed silylation as used in this work cannot be utilized as a persilylation procedure as presented by Sakauchi and Horning ( 4 ) or Aringer e t al. (5). The most interesting feature of this reaction might be its use for easily obtaining a good gas phase derivative for the study of thermally unstable steroid structures, primarily the dihydroxy acetone side chain. Several derivatives have been introduced in the past (10, 12, 13) and from a quantitative point of view, the double type derivatives ( e . g . , methoxime-TMS) appeared the most satisfactory (14-16). In all cases with this type of compound, the TMS-enol-TMS derivative offered a single product with satisfactory gas phase behavior. In the case of 3-keto-4-ene structure, after hydrolysis of the 3-enol-TMS (evaporation and redissolution in HMDS), the final 3-ketonic product was contaminated by 1 to 4% of the remaining 3-enol-
TMS. The proposed structure of the TMS-enol-TMS stabilizing the dihydroxy acetone side chain could be questioned since a base catalyzed D-homo rearrangement might occur R. V. Kelly, Steroids, 13, 507 (1969). G. M. Anthony, C. J. W. Brooks, J. MacLean, and I. Sangster, J . Chromatogr. Sci., 7, 623 (1969). W. L. Gardiner and E. C. Horning, Biochem. Biophys Acta. 115, 524 (1966). P. G. Devauxand E. C. Horning.Anab Lett., 4, 151 (1971). T. A . Baillie, C. J. W . Brooks, and E. C. Horning, personal communication, 1972.
I
501
-.
"a
&-
//
1
2
"
20
HOURS Figure 10. Reaction of ESTFA (upper) and BSA (lower) with androsterone in the presence of potassium acetate. (Fr: free steroid; 3 T-17 K: 3 mono TMS derivative; 3 T-17 eT: 3 TMS, 17enol-TMS product)
(17). However, a study of the acid and base hydrolysis product of the TMS-enol-TMS for T H E never showed evidence for compounds other than the starting steroid as analyzed by GLC as MO-TMS derivative. The NMR spectrum for the cortisone derivative gave final evidence for a side chain remaining in place, with the introduction of a C-20-C-21 double bond, and 3-TMS groups in 17a, 20 and 21. The mechanism of the enol-trimethylsilyloxy formation under base catalysis from ketonic nonsteroidal compounds has been studied in detail by House et al. (18) and shown to involve in a first step the formation of an enolate ( e . g . , in pyridine-triethylamine). I t was concluded that a proton transferring agent was necessary and this is confirmed by the fact that anhydrous sodium (or potassium) acetate was not effective in our hands, whereas the crude, hydrated reagent always gave a high yield of enol product. However, we found that mixing the nucleophilic and the silylating agents was more effective and convenient for the structures studied. The practical applicability of this derivative has been investigated in a preliminary study of the saturated corticosteroid metabolites in human urine, after isolation of a corticosteroid fraction by silycic acid column chromatography. Satisfactory quantitative and qualitative results were obtained. Figure 11 shows the gas chromatographic analysis as TMS-enol-TMS of the polar fraction obtained after florisil column chromatography of a crude urinary steroid extract. This approach makes possible a rapid isolation procedure for the main corticosteroid metabolites, D.N. Kirk and M. P. Hartshorn, in "Steroid Reaction Mechanism," Elsevier, Amsterdam, 1968, p 294. (18) H. 0. House, L. J. Czuba, M. Gall, and H. D. Olmstead. J , Org. Chem.. 34, 2324 (1969). (17)
ANALYTICAL C H E M I S T R Y , VOL. 45, NO. 7, JUNE 1973
1097
OV 101 1V. 121,. 200-290 x1.2 Wmln
URINEGON. FLORISIL 3
,I
TMSmol TMS
1
Figure 11. Gas chromatographic analysis of the florisil column chromatography fraction containing the major urinary corticosteroid metabolites. C. B.: cholesterol butyrate which might be of interest for example for the estimation of secretion rates in vivo. The application to complex biological mixtures, such as a total neutral urinary extract, would however yield some undesirable enol-TMS by-products, particularly from the 17-keto steroids. This might be minimized by transfer in another solvent but further study remains to be done as far as the quantitative aspects are concerned. In conclusion, base-catalyzed silylation offers a procedure to form TMS-enol-TMS from hydroxy keto steroids. The base-catalyzed reaction appears totally different from the usual acid silylation catalysis with regard to the reactivity of the different keto groups of the steroids of hormonal interest. This derivative is most easily formed from
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A N A L Y T I C A L C H E M I S T R Y , VOL. 45, N O . 7, JUNE 1973
a ketolic or a dihydroxy acetonic side chain, being a new and convenient way of studying corticosteroid metabolites by gas phase methodology.
ACKNOWLEDGMENT We are indebted to C . J. W. Brooks for mass spectrometry analysis and helpful discussion throughout this work. Received for review November 30, 1972. Accepted January 29, 1973. This work was made possible thanks to the financial support from the Di!lCgation GCnkrale B la Recherche Scientifique et Technique, the Institut National de la Santi! et de la Recherche MBdicale, and the Fondation pour la Recherche Mbdicale.
