tert-Butyl Dimethylsilyl Ethers as Derivatives for Qualitative Analysis of Steroids and Prostaglandins by Gas Phase Methods R. W. Kelly" and P. L. Taylor Medical Research Council, Unit of Reproductive Biology, 2 Forrest Road, Edinburgh, EH 1 2Q W, Scotland
An improved method of preparation of fed-butyl dimethylsilyl (f-BDMS) ethers of steroids and prostaglandins is reported. When this technique is used, good yields of derivative are obtained even at low levels and the range of mixed derivatives for GCMS is extended. M 57 is almost always the base peak in f-BDMS spectra of steroids and prostaglandins and the few exceptions (including one influenced by instrumental condltlons) are reported.
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The choice of derivative is a critical step in gas chromatography (GC) and gas chromatography-mass spectrometry (GCMS) but the range of suitable derivatives is restricted. We report here an improved method of preparing tertbutyl dimethylsilyl ( t -BDMS) ethers which extends their utility and scope in gas phase analysis. The new method is a modification of that reported by Corey and Venkateswarlu ( I ) . This technique (unlike the original method) gives high yields of submicrogram quantities of steroids and prostaglandins. Because a nonaqueous isolation technique is used, mixed derivatives with easily hydrolyzable groups can also be synthesized. The main advantages of t -BDMS ethers are their simplified mass spectra, which usually have their base peak a t M - 57, and their stability towards hydrolysis. t-BDMS ethers have properties complementary to those of T M S ethers in these respects, because T M S ethers are lo4 times less stable ( 2 ) and the spectra of T M S ethers show a variety of fragmentation pathways and they sometimes lack an appreciable molecular ion.
EXPERIMENTAL Derivative Formation. t-Butyl Dinethylsilylation. Samples were evaporated a t the bottom of a 2 X 60 mm glass tube, 50 pl of imidazole (2 M in dry DMF) and 50 pl of t-butyl dimethylsilyl chloride ( 2 M in DMF, from Willowbrook Laboratories, or synthesized according to Ref. 1) were added, and the tube was sealed and heated a t 100 O C for 1 hr. The contents were placed on top of a 0.5 X 3 cm column of Sephadex LH20 swollen in heptane/ethyl acetate ( 3 : l ) . The column was eluted with 4 ml of the same solvent and the eluate evaporated to dryness. Cyclic Boronates. The sample was dried in a 2 X 60 mm glass tube and 35 p1 of a solution of alkyl boronic acid (5 mg/ml in acetone/benzene 2 1 ) was added. The solvent was evaporated a t 60 O C in vacuo over a period of 30 min. Methyl Esters. These were formed by treatment for 5 min with diazomethane in ether/methanol. T M S Ethers. These were made with bl's(trimethylsily1)trifluoro acetamide. Oximes. The sample to be oximated was dissolved in a small quantity of ethanol and added to 5 ml alkoxyamine hydrochloride (10mg/ml) or hydroxylamine hydrochloride (20 mg/ml) in aqueous pyridinium acetate (pH 5 , 1.5 M). The mixture was placed in an ultrasonic bath a t 60 OC for 40 min, then extracted with ether/ ethyl acetate (3:l). The ethereal layer was washed and evaporated to dryness. Gas Chromatography Mass Spectrometry. Most t-BDMS spectra were recorded on an A.E.I. MS 12 mass spectrometer coupled through a Watson-Biemann separator to a Hewlett-Packard 402 gas chromatograph. Data acquisition was through an A.E.I. DS20 system. The GC columns were 2 X 1500 mm made of glass
and with glass-to-metal seals. They were packed with 1.5% Dexsil 300 on 100/120 mesh Chromosorb G. Carrier gas flow was 20 ml per minute. Lines connecting the GC to the separator were glasslined stainless steel (S.G.E. Ltd.) and maintained a t 300 "C. The separator was kept a t 320 OC and the glass line to the source was a t 280 "C. Source temperature was 260 "C. Ionizing voltage was 20 eV and trap current was 100 p A . Some spectra were recorded on a Du Pont 490B spectrometer coupled through an all glass jet separator to a Varian 1400 GC with a support coated open tubular column (S.G.E. Ltd.). In this instrument, spectra were recorded on a UV oscillograph.
RESULTS AND DISCUSSION The improvement in the preparation of t-BDMS derivatives is by filtering the reaction mixture through Sephadex LH20, thus avoiding partial hydrolysis during an aqueous work-up. The filtering of solutions through lipophilic Sephadex has been used elsewhere to purify T M S and oxime derivatives ( 3 ) , but this technique has not been applied previously to t-BDMS derivatives. We have found that it simplifies the preparation and expands the uses of these derivatives. Using this technique, we can obtain quantitative yields of t-BDMS ethers of heavily oxygenated compounds such as prostaglandins and also prepare a wide range of t-BDMS derivatives. Both of these advances are valuable in qualitative analysis, allowing a wider application of these derivatives in GC and GCMS. The distinguishing feature in the mass spectrum of a t BDMS derivative is the strong M - 57 peak due to the ready loss of a t-butyl radical; an example is shown in Figure 1. We have made a study of a wide range of t-BDMS derivatives of steroids and prostaglandins, and it is only the exceptional spectrum which does not have M - 57 as the base peak. The exceptions to M - 57 as a base peak are informative as well as being important in qualitative analysis. The formation of t-BDMS ethers appreciably increases the retention times of steroids and prostaglandins. The relevant retention data of several steroids and prostaglandin are shown in Table I. Steroids. The mass spectra of t-BDMS ethers and the t-BDMS oximes of steroids show almost universal M - 57 base peaks which are useful for identification and quantitation. An examination of possible derivatives of progesterone exemplifies one of the uses of t-BDMS derivatives of steroids. A comparison of the T M S and t-BDMS oximes of progesterone is shown in Figure 2; the dramatic simplifica-
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Figure 1. Mass spectrum of 3b-hydroxyandrost-5-en- 17-One (dehydroepiandrosterone) t-BDMS ether ANALYTICAL CHEMISTRY, VOL. 48, NO. 3, MARCH 1976
465
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Other steroids which show strong molecular ions are the estrogens, where M - 57 is the base peak for estradiol but the molecular ion is almost as strong. In the case of the corresponding TMS compounds, large molecular ions are also encountered ( 4 ) . Where the steroid contains other labile groups such as underivatized hydroxyls or close oxygen functions such as in cu-ketols, then elimination reactions a t the site will compete with the loss of the butyl radical from the t-BDMS derivative. In some cases, this will result in other peaks equaling the M - 57 peak. Examples are 3p(t-butyl dimethylsilyloxy), Ilp-hydroxyandrost-5-en-17one with a base peak a t M - 57 [but M - (57 18) being 80%of base peak] and 3P,lGP-di(t-butyldimethylsi1oxy)androst-5-en-17-one with a base peak a t M - (132 57). Prostaglandins. In the prostaglandin field, the t-BDMS compounds which do not have M - 57 as their base peak are mixed derivatives containing acetates or other silyl groups. Also, it is probable that enol ethers of prostaglan-
~~
Table I. Retention Data Carbon No. Carbon No. as T M S as t.BDMS Compound
ether
ether
3fl-Hydroxyandrost-5-en17-one (DHA)
27.1
29.6
Prostaglandin F,, methylester (tris derivative) Prostaglandin E, methylester methyl oxime (bis derivative)
26.5
32.7
26.8
31.0
+ +
tion typifies the changes which are brought about by the transition TMS to t-BDMS. Progesterone can also be made into the t-BDMS enol ether, and this compound has the molecular ion as the base peak, Figure 2. This is an example of stabilizing the molecular ion by delocalization of the charge.
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Figure 2. Comparison of the TMS oxime and the f-BDMS oxime of pregn-4-ene-3,20-dione (progesterone) Above is the enol CBDMS of progesterone with the molecular ion (m/e 428) as the base peak
466
ANALYTICAL CHEMISTRY, VOL. 48, NO. 3, MARCH 1976
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Figure 3. Mass spectrum of 9a,11a,15,19-tetrahydroxyprosta-5,13dienoic acid (19-hydroxyprostaglandin F2J as methyl ester, tetra-t-BDMS ether (upper spectrum) and as methyl ester tetra-TMS ether (lower spectrum)
dins would have the molecular ion as the base peak due to the stabilization of this ion by charge delocalization. In the case of the methyl ester, t-BDMS ether of PGB, which has already been reported ( 5 ) , extended conjugation gives rise to the stabilization of the molecular ion and its consequent high abundance although M - 57 is still the base peak. Initially, on the MS12 instrument, prostaglandin Fzo, methyl ester, t-BDMS ether gave a base peak at M - (132 57) due to the loss of t-butyl silanol as well as a butyl radical and in some spectra M - 57 was absent. However, when the spectrum of the same derivative was examined a t the high mass end on a different GCMS combination (Du Pont 490), the M - 57 peak was the base peak. It is possible that the loss of 132 mass units is induced by thermal processes rather than by 'electron impact. Thus, instrumental factors can drastically change a t-BDMS spectrum, and this should be remembered when inter-laboratory comparisons are made. Apart from the exceptions mentioned above, every naturally occurring prostaglandin as t-BDMS oxime, ester, or ether gives an M - 57 base peak. This is not always the case for mixed derivatives. Where butyl boronates are combined with t-BDMS derivatives, the dominant effect is the ionization a t the silyl group and M - 57 is the base peak. However, when t-BDMS groups are introduced by exchange with TMS ethers or esters and a mixed TMSItBDMS compound results (e.g., E2 methyl oxime, TMS ether, t-BDMS ester), then ionization is apparently equally spread among silyl groups and features of TMS spectra dominate. This new preparation technique also allows selective reaction a t the 9 and ll positions in prostaglandin Fze by first forming the methyl ester, butyl boronate, making a t-BDMS ether of the 15 hydroxyl, hydrolyzing the butyl boronate in ethanol and converting the free hydroxyls to derivatives such as T M S ethers. The spectrum of such a compound (PGF2, methyl ester, 15-t-BDMS, 9 , l l TMS ether) has a base peak at mle 237 (M - 57 is 83% base peak). The use of TMS oximes for steroids has already been described (6) but t-BDMS oximes have not so far been re-
+
ported, although they extend to ketonic compounds the advantages of the t -BDMS ethers. When ketonic compounds such as prostaglandin E2 are made into t-BDMS oximeltBDMS ethers, the mass spectrum is simplified because competitive ionization a t the alkyl oxime is eliminated. An example of the value of t -BDMS derivatives from our laboratory is the recent identification of 19-hydroxyprostaglandin Fza (9a,lla,l5,19-tetrahydroxyprosta-5,13-dienoic acid) (7); this compound was found by searching for more polar compounds following the discovery of the 19-hydroxyprostaglandins E (8, 9). In the case of the 19-hydroxyprostaglandin Fza, one is left with doubt of identity after examining the methyl ester TMS spectrum because the highest peak is a t mle 582 (M - 90) and although other evidence points to the extra hydroxyl, the t-BDMS spectrum has a base peak at m/e 785 (Figure 3) which leaves no doubt of the extra hydroxyl.
ACKNOWLEDGMENT We are grateful to N. S. Crossley of I.C.I. (Pharmaceuticals) Ltd., for the supply of 19-hydroxyprostaglandin Fza and to J. E. Pike of the Upjohn Company for the supply of prostaglandins E and F.
LITERATURE CITED (1) E. J. Corey and A. Venkateswarlu, J. Am. Chern. SOC.,94, 3190 (1972). (2) L. H. Sommer, "Stereochemistry, Mechanism and Silicon", McGraw Hill, New York, N.Y., 1965, p 138. (3) M.Axelson and J. Sjoval. J. SteroidBiochern., 5, 733 (1974). (4) T. Luukkainen and H. Aldercreutz, Biochern. Biophys. Acta, 107, 579 (1965). (5) J. Throck Watson and 8. J. Sweetman, Org. Mass Spectrorn., 9, 39 (1974). (6) C. J. W. Brooks, E. M. Chambaz, W. L. Gardiner and E. C. Horning, Proceedings of the second international congress on Hormonal Steroids, Milan, 1966, Excerpta Medica, p 366. (7) P. L. Taylor and R . W. Kelly, FEBS Lett., 57, 22 (1975). (8) P. L. Taylor and R. W. Kelly, Nature (London), 250, 665 (1974). (9) H. T. Jonsson, 6. S. Middleditch, and D. M. Desiderio, Science, 187, 1093 (1975).
RECEIVEDfor review August 8, 1975. Accepted November 24, 1975.
ANALYTICAL CHEMISTRY, VOL. 48, NO. 3, MARCH 1976
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