Steroids, trimethylsilyl derivatives

In the steroid field, it has been shown that O-trimethylsilyl derivatives may be converted directly into trifluoroacetates,192,193 and the relative merits of these two derivatives for the separation of glycols have been discussed.194... 

Trimethylsilyl derivatives for the analysis of steroids were first described by Luuk-kainen et al. [316]. HMDS in tetrahydrofuran with the addition of TMCS as a catalyst... 

In the human female, whose pituitary hormones may be suppressed by the use of oral contraceptives, the ratio of testosterone to creatinine in urine may be used. Alternatively, a method suitable for both males and females is to measure the ratio of total testosterone to epitestosterone using gas chromatography—mass spectrometry. In this method, the bis(trimethylsilyl) derivative is formed and the intensity of the molecular ions at m/z 432, under electron impact conditions for both of the steroid derivatives, is used to determine the ratio (M. Donike and J. Zimmermann, J. Chromat, 1980, 202, 483-486). The internationally accepted limit for the ratio is currently 6. 

The sensitivity of Si chemical shifts to structural changes and the technique of silylating compounds for more favourable analysis or synthesis have been combined by several researchers to produce a powerful structure elucidation technique for monofunctional or polyfunctional compounds. (135-141) Specifically, the trimethylsilyl derivatives of imidophosphoryl compounds, (141) sugars, (138-140) steroids, (140) amines, amides, and urethanes, (135,136) and amino-, hydroxy-, and mercaptocarboxylic acids (137) have all been studied within the past three years. 

The collection of carbon-14 and tritium-labeled steroids in GLC has been described by Kliman and Briefer (K8) with application to the analysis of testosterone in human plasma. Using the phenylmethylsiloxane polymer, OV-17, as liquid phase, Homing (H13) has achieved complete separation of testosterone and epitestosterone, both as free steroids and as trimethylsilyl derivatives. Figure 5 illustrates these separations. 

Fig. 10. GLC of trimethylsilyl derivatives of synthetic steroid mixture. (A) Procedure using 2% neopentyl glycol succinate on Chromosorb W, acid-washed, dimethyldichlorosilane treated, 80-100 mesh, in 6 foot X 5 mm i.d. glass column at 215° with 10 psi nitrogen. (B) Procedure using 35% XE-60 on Anakrom ABS (90-100 mesh), in 6 foot X 5 mm i.d. glass column, at 232°, with 5 psi nitrogen. Reproduced from Ruchelman and Cole (R4) with permission.

As an example of a clear improvement in separation we can cite the data on the retention of steroids on a non-polar silicone stationary phase, SE-30, as presented by Heftmann [54]. Two monohydroxy-steroids, 5a-cholestan-3 3-ol and 5-cholesten-3 3-ol, have the same relative retention (2.85) on a column containing a non-polar stationary phase (internal standard cholestane), but the relative retentions of their trimethylsilyl derivatives are 2.60 and 2.55 and those of their chlorodichloroacetates are 3.79 and 3.62, respectively. The relative retention times of 3a-hydroxy-5a-androstan-17-one and 3j3-hydroxy-5a-androstan-17-one are similar at 0.96 and 1.00, respectively, whereas those of their TMS ethers are 0.46 and 0.61, respectively. 

For one, the mass spectra of trimethylsilyl derivatives may not be suitably informative. Frequently, the intensities of molecular-ion and useful fragment peaks are low compared with those of the ubiquitous MejSi m/e = 13), MejSiOH (m/e = 15), and MejSiOSiMej" (w/e = 147) peaks, obscuring clues as to the identity of low concentration components. Polyhydroxy compounds, such as certain steroids, tend to undergo fragmentation by successive losses of trimethylsilanol (MejSiOH) molecules, exhibiting series of peaks 90 mass units apart, with attendant uncertainty as to whether the molecular-ion peak is the highest-mass member of the series or something invisible beyond that. 

Whereas the earlier work used GC for the estimation of only selected urinary steroid constituents, most current work favors the multicomponent (metabolic profiling) approach. The preparation of mixed derivatives , such as, for example, the methoxime-trimethylsilyl derivatives of Gardiner and Homing [291], in principle facilitates conversion of all metabolites containing hydroxy and carbonyl groups. Through the introduction of capillary columns to steroid analysis [12,292,293], it became possible to separate the complex mixtures of such derivatives. 

Alternatively, the concentration of BHA was increased to 50 mg ml in dry pyridine and the O-benzyloxime was silylated with N-trimethylsilylimidazole (TMSI) at 150-160 °C for 3h. GC-MS showed that the major metabolites of the adrenocortical steroid hormones were converted into O-benzyloxime and trimethylsilyl derivatives. 

O-Methyloxime—trimethylsilyl derivatives of steroids from urine [61]... 

The great dilemma of the GC analysis of steroids is whether to use derivatization or not. In the case of thermally unstable steroids, the answer is unequivocally yes. Steroids of this type are mainly the corticosteroids, containing the unstable a-ketol side chain. Of several possibilities, double derivatization to form the methoxime trimethylsilyl derivatives is the most widely used (see reaction [I]) ... 

Figure 1 GC-MS Profile analysis. Total ion chromatogram profiles produced by scanning methoxy-trimethylsilyl derivatives of urinary steroids. (A) Profile from a patient with 11 -hydroxylase deficiency in which the principal steroids are metabolites of IIjS-deoxycortisol (substance S) such as tetrahydrosubstance S (THS) and hexahydrosubstance S epimers (HHS). (B) Profile from the patient s father this profile is essentially normal. Some of the major metabolites are C19 steroids such as androsterone (An), et-iocholanone (Etio), pregnanetriol (PT), and cortisol metabolites (THE, tetrahydrocortisone THF, tetrahydrocortisol and 5a-THF, 5a-tetrahydrocortisol). Peaks 1-3 are internal standards. (Reprinted from Shackleton CHL, Merdinck J, and Lawson AM (1990) In McEwan CN and Larsen BS (eds.) Mass Spectrometry of Biological Materials, pp. 297-377. New York Dekker courtesy of Marcel Dekker Inc.)...

After considerable research, which examined a wide variety of different derivatives, trimethylsilylation has emerged as the procedure of popular choice for steroid and steroidlike compounds. Pure compounds or biological extracts containing 3 (3-hydroxyl groups can be readily silylated by treatment with A,G-bis-trimethylsilyltrifluoro-acetamide containing 1 % trimethylchlorosilane at 60°C for 30 min, with or without added pyridine. A,G-bis-tri-methylsilyltrifluoroacetamide, under some circumstances, can also be used alone or with a mixture of hexamethyl-disilazane and trimethylchlorosilane (10 10 5 v/v/v) at 60°C for 30-60 min. The trimethylsilyl derivatives separate well on capillary columns carrying apolar stationary phases. The derivatives also provide excellent electron-impact mass spectra. 

Harvey, DJ., and Vouros, R (1979) Influence of the 6-trimethylsilyl group on the fragmentation of the trimethylsilyl derivatives of some 6-hydroxy-and 3,6-dihydroxy-steroids and related compounds. Biomedical Mass Spectrometry, 6,135-143. 

Vouros, R, and Harvey, D.J. (1980) The electron impact induced cleavage of the C-17-C-20 bond D-ring in trimethylsilyl derivatives of C21 steroids. Reciprocal exchange of trimethylsilyl groups. Biomedical Mass Spectrometry, 1,217-225. 

Harvey, D.J., Homing, M.G., and Vouros, P. (1971) Some stereochemical factors in the formation of rearrangement ions in the mass spectra of trimethylsilyl derivatives of steroidal phosphates. Tetrahedron, 27,4231-4243. 

Conversion of A -3-ketosteroids or their trimethylsilyl or acetyl derivatives in fluorescent components, whereby the detection limits were improved by 65% for the acetates. A -3-keto- and A -3-OH-steroids also react with the same sensitivity. 

System (5) has been used for the direct analysis of steroids, including cortisone and cortisone acetates, as the trimethylsilyl ethers of their methoxyimmino derivatives [144], A glass column (180 cm x 4 mm) containing 1.5% SE-30 on 60 to 80 mesh Chromosorb W, operated at 235°C, is used for the separation. Nitrogen is used as the carrier gas (flow rate of 35 mL/min), and detection is by flame ionization. 

Chemical derivatization is a standard procedure for GC-MS analysis of steroid hormones, because steroid hormones are not volatile to go through GC column [1, 6], The major concerns of derivatization reagents for GC-MS analysis of steroid hormones are the completeness of the derivatization reaction and the volatility of hormone derivatives. The typical derivatization reagents for GC-MS samples are silylation reagents, e.g., Af-methyl-Af-trifluorotrimethyl acetamide (MSTFA, [37, 39]) and A,0-bis(trimethylsilyl) trifluoroacetamide (BSTFA, [33]), which react with both alcoholic and phenolic hydroxy groups on steroid hormone molecules. 

Suitable derivatives to render most steroids more stable and volatile for gas chromatography and to improve their mass spectrometric properties have been developed. The trimethylsilyl ethers [206] or the methoxime-trimethylsilyl ethers of hydroxy keto steroids [207] have been widely used and their mass spectra extensively studied [204], A comparison of the GC-MS behaviour of several derivatives of some of the adrenocortical hormones has been made [208], including methoxime-trimethylsilyl ethers, dimethylsiliconides, methyl boronates, oxetanones and acetonides. The recently described improved preparation of enol-trimethylsilyl ethers of corticoids [209] should also prove of value. 

Ring-opening reactions of cyclopropanone hemiketals are well known. Under appropriate conditions, cleavage of trimethylsilyl protected hemiketals can provide a synthetically useful route to homoenolate anions as noted earlier (Scheme 20). The reaction of an isopropoxy-titanium homoenolate (128) derived from 127 with an aldehyde has recently been used as the key step in the stereocontrolled construction of the steroidal side-chain of depresosterol (Scheme 49) ... 

New derivatives of steroidal hormones, prepared for biological evaluation, include 17-dialkylaminoalkanoates and other novel esters of 17a-hy-droxyprogesterone," trimethylsilyl ethers of 17a-hydroxyprogesterone, testosterone, 19-nortestosterone, 17a-methyltestosterone, and other androstane... 

---