Method for Selective Introduction of Trimethylsilyl and Perdeuterotrimethylsilyl Groups in Hydroxy Steroids and Its Utility in Mass Spectrometric Interpretations Paul Vouros and D. J. Harvey Institute f o r Lipid Research, Baylor College o f Medicine, Houston, Texas 77025 A method is described for the preparation of mixed trimethylsilyl (TMS) and perdeuterotrimethylsilyl (TMS-
Sa-androstane-3~,11P-diol-17-one methyloxime (III), 5apregnane-3/3,17a-diol-20-one methyloxime (IV), 5-pregnene-
do)derivatives of hydroxy steroids in which the TMS-ds group occupies a specific position. This method of selective silylation is based on the different silylation rates of sterically hindered and unhindered hydroxyl groups. Examples are given showing the utility of this method of derivatization in the interpretation of the mass spectra of trimethylsilyl derivatives of hydroxy steroids.
3P,17a-diol-20-one methyloxime (V), 17a-methyl-5a-androstane-3@,17P-diol (VI), 17a-methyl-5-androstene-3P,17P-diol (VII), and 17a-ethyl-5-androstene-3P,17P-diol (VIII). Their structural formulas as well as those of their trimethylsilyl and perdeuterotrimethylsilyl derivatives are shown below. All compounds contained a sterically hindered hydroxyl group (llp-hydroxy in 1-111; 17a-hydroxy in IV and V, and 17phydroxy in VI-VIII) whose resistance to silylation by many silylating reagents has been well established (8-11).
INTERPRETATION OF THE MASS SPECTRA of the trimethylsilyl (TMS) derivatives of numerous compounds has been greatly facilitated by the introduction of perdeuterotrirnethylsilylating reagents (I). Subsequently, the use of mixed TMS and perdeuterotrimethylsilyl (TMS-dg) derivatives has been described, in which the TMS-dg group occupies a specific position (2-5). The preparation of these compounds was accomplished by utilizing the higher lability of acidic as compared with ethereal TMS groups in exchange reactions on a gas chromatographic column connected to the mass spectrometer. McCloskey and coworkers have also employed randomly mixed trimethylsilyl and perdeuterotrimethylsilyl derivatives prepared from an equimolar mixture of labeled and unlabeled silylating reagents, to determine the structures of doubly charged ions observed in the mass spectra of trimethylsilylated pyrimidines and purines (6). Selective labeling of the trimethylsilyl groups should be particularly helpful in the interpretation of the mass spectral fragmentation of the TMS derivatives of hydroxy steroids, since the mass spectra of these compounds are often dominated by structurally informative trimethylsilyl-containing ions (7). We have examined the mass spectra of the TMS derivatives of several hydroxy steroids by selectively introducing TMS-dg groups in certain positions. Selective silylation was accomplished by utilizing the different rates of reaction of sterically hindered and unhindered hydroxyl groups towards various trimethylsilylating reagents (8-10). The steroids chosen for this investigation were Sa-androstane-30,ll P,17p-triol (I), 5-androstene-3P,11~,17~-triol (11),
H
I
ORZ
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(1) J. A. McCloskey, R. N. Stillwell, and A. M. Lawson, ANAL. CHEM., 40, 233 (1968). (2) A. M. Lawson, R. N. Stillwell, M. M. Tacker, K. Tsuboyama, and J. A. McCloskey, J . Amer. Chem. Sac., 93,1014 (1971). (3) D. J. Harvey, M. G. Horning, and P. Vouros, Tetralzedrorz,27, 4231 (1971). (4) D. J. Harvey, M. G. Horning, and P. Vouros, J. Cliem. SOC., Perkin Trans. I , 1972, 1074.
( 5 ) B. S. Middleditch and D. M. Desiderio, Baylor College of Medicine, Houston, Texas, unpublished data, 1972. ( 6 ) E. White, V, P. M. Krueger, and J. A. McCloskey, J . Org. Chem., 37, 430 (1972). (7) J. Diekman and C. Djerassi, ibid., 32, 1005 (1967). (8) E. M. Chambaz and E. C. Homing, Anal. Lett., 1, 201 (1967). (9) E. M. Chambaz and E. C . Homing, Anal. Biocliem., 30, 7 (1969). (10) E. Sakauchi and E. C. Homing, A/ral. Lett., 4, 41 (1971).
m
IIt
H3
H
m
Rlo&~ti~-CH3
R 10
rn
-sm
111-VIII(R' R 2 = H) IIIa-VIIIa(R1 = R2 = TMS) IIIb-VIIIb(R1 = R* = TMS-dn) IIIc-VIIIC(R' = TMS; R2 = TMS-do)
(11) J-P. Thenot and E. C. Horning, Anal. Letr., 5 , 21 (1972). ANALYTICAL CHEMISTRY, VOL. 45, NO. 1, JANUARY 1973
7
Table I. Partial Mass Spectra and Mass Shifts of the Most Significant TMS-Containing Ions in the Mass Spectra of 1Ia-c
129 [100p 143 1841 169 [77] 252 [63] 303 1371 327 [71] 342 [62] 391 [14] 417 [41]
138 152 178 252 312 336 351 409 432 (2)b 435 (3)b 450
129 152 169 252 303 327 342 400 417 (5) 426 (2) 432 [92] 432 (1) 441 (1) 522 [24] 549 531 Figures in brackets refer to the relative intensity of the ion of indicated mass. * Figures in parentheses refer to the ratios of the indicated peaks. 0
Table 11. Partial Mass Spectra and Mass Shifts of the Most Significant TMS-ContainingIons in the Mass Spectra of IIIa-c IIIa (M = 479) IIIb IIIC [3~,11~(3P-TMS-do; [3P,llP(TMS-dgM 11P-TMS-dg) (TMS-dohl 143 [6p 152 152 169 [41 178 178 191 182 [l2] 191 207 198 181 207 213 ['GI 222 222 268 [25] 268 268 358 [36] 367 358 (9) 367 (1) 448 [100] 466 457 464 [6] 479 (1)b 473 482 (5)* 479 [8] 497 488 Figures in brackets refer to the relative intensity of the peak. b Figures in parentheses refer to the ratios of the indicated peaks. 0
EXPERIMENTAL Apparatus. Mass spectra were recorded with an LKB-9000 mass spectrometer. The electron energy was 70 eV, ion accelerating voltage 3.5 kV, and source temperature 250 "C. Samples were introduced aia the gas chromatographic inlet employing a 6-ft, 1 OV-17 column temperature programmed at 4"/min from 180 "C. Reagents. Hydroxy steroids were obtained from commercial sources. N,N-Bis(trimethylsily1)trifluoroacetamide (BSTFA), hexamethyldisilazane (HMDS), and trimethylsilylimidazole (TSIM) were obtained from Pierce Chemical Company. Perdeuterotrimethylsilylimidazole (TSIM-d,) was obtained from Merck Laboratory Chemicals. Procedure. Fully silylated TMS-do and TMS-d, derivatives were prepared by standard techniques. PREPARATION OF SELECTIVELY LABELED DERIVATIVES Of I, 11, VI, VII, and VIII. The hydroxy steroid (0.5 mg) was dissolved in anhydrous acetonitrile (50 pl) and BSTFA (15 pl). The solution was heated at 60 "C for 1hr and was then allowed to stand at room temperature overnight. This resulted in virtually complete silylation of the unhindered hydroxyl groups (3p,17p in I and 11; 36 in VI, VII, and VIII). The samples were blown to dryness with nitrogen at room temperature. The residue was redissolved in cyclohexane (50 pl) and TSIM-d9 (15 11.) and the solution was heated at 60 "C. Aliquots were taken at 30-min intervals and examined by combined gas chromatography-mass spectrometry, to de8
ANALYTICAL CHEMISTRY, VOL. 45, NO. 1, JANUARY 1973
termine both the extent of reaction and the amount of exchange between trimethylsilyl and perdeuterotrimethylsilyl groups from the reagent. PREPARATION OF SELECTIVELY LABELEDDERIVATIVES OF 111-V. The methyloxime derivatives were prepared as described previously (12), extracted with ethyl acetate and, after removal of the solvent, the sample was redissolved in acetonitrile (50 pl) and HMDS and allowed to react overnight at room temperature. The perdeuterotrimethylsilyl group was introduced to the hindered positions by reacting the compound with TSIM-ds as described above. RESULTS AND DISCUSSION Methodology. The method used to introduce labeled trimethylsilyl groups into a specific position involved: (i) the silylation of unhindered hydroxyl groups under mild conditions; (ii) removal of solvent and reagents; and (iii) reaction of the remaining hydroxyl groups under more severe conditions with deuterium labeled silylating reagents. By the use of carefully controlled experimental conditions, it was possible to prepare derivatives in which the perdeuterotrimethylsilyl group occupied the hindered position with a specificity of 90% or more. The main difficulties which had to be overcome in accomplishing this were: (a) prevention of any silylation of the hindered hydroxyl groups during the first step of the reaction, but at the same time ensuring complete silylation of the unhindered hydroxyl functions ; (b) prevention of hydrolysis of the TMS ethers before introduction of the TMS-d9 group; and (c) catalytic exchange of unlabeled TMS and TMS-dggroups. The problems involved in the initial silylation step were minimized by using only mild silylating reagents (9, 10) (no trimethylchlorosilane or other catalyst was used at this stage) and by monitoring the reaction with gas chromatographymass spectrometry. The reaction was stopped by cooling the mixture to -10 "C when the desired degree of silylation had been achieved. Hydrolysis during the removal of the solvent and reagents was minimized by using well dried glassware, nitrogen, reagents, and solvent. Volatile reagents (HMDS or BSTFA) and solvent (CH8CN) were selected during the first step to facilitate their removal. Cyclohexane was found to be the best solvent for the second silylation step. Other solvents (e.g., CH,CN, pyridine) were found to cause some hydrolysis of trimethylsilyl ethers and the consequent replacement of these groups with labeled trimethylsilyl groups. The need for complete removal of the original silylating reagents is thus apparent. In the case of the methyloxime derivatives, it was necessary to remove all traces of salts by extracting the sample with ethyl acetate. The ethyl acetate layer was then dried with magnesium sulfate to remove any excess moisture. It was often necessary to sacrifice the yield of fully silylated derivative in order to achieve optimum purity of selectively labeled steroid. However, the derivatized steroids were easily separated from the free hydroxy steroids during GCMS. Mass Spectrometric Interpretations. Compound I was used to assess the specificity of the labeling procedure because the mass spectrum of its trimethylsilyl derivative has been studied in detail by means of both deuterium and l*O-labeling as well as high resolution mass spectrometry (13). The spectrum of this derivative exhibited abundant trimethylsilyl(12) C. J. W. Brooks and D. J. Harvey, Steroids, 15, 283 (1970). (13) P. Vouros and D. J. Harvey, 20th Annual Conference on Mass Spectrometry and Allied Topics, Dallas, Texas, June, 1972, paper No. A7.
I
73 6.1
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Figure 1. Top: Mass spectrum of 3p,17~~-bis(trimethylsilyloxy-d~) 5-pregnen-20-onemethyloxime (M = 505) Center: Mass spectrum of 3p,l7~~-bis(trimethylsilyloxy-d~) 5-pregnen-20-onemethyloxime (M = 523) Bottom: Mass spectrum of 3p-(trimethylsilyloxy-d~)-17~~-(trirnethylsilyloxy-d~)-5-pregnen-2O-one methyloxime (M = 514). The peak at mje 523 represents the partial exchange of the 3p-(TMS-do)group with TMS-ds from the reagent
containing ions at mje 143, rnje 169, mje 182, mie 191, and m/e 196. The origin of these ions has been established (13) and the data obtained from the spectrum of the tris-TMS ether, selectively labeled with one TMS-dg group in the l l p position (IC),are in agreement with the earlier results. The identities of the TMS-containing ions in the mass spectra of two other 11P-hydroxylated steroids (11, 111) were examined by the selective TMS-labeling method. The partial mass spectra of their trimethylsilyl and perdeuterotrimethylsilyl derivatives, as well as the derivatives which were selectively labeled with a TMS-dggroup on the 1lP position (IIaIIc, IIIa-IIIc) are given in Tables I and 11. The TMS-containing ions are readily apparent from their nine, eighteen, or twenty-seven amu shifts exhibited in the spectra of IIb and IIIb. The ion at m/e 129 in IIa, characteristic of A5-olefinic steroids (7, 14-16) containing a 3-trimethylsilyloxy group, (14) P. Eneroth, K. Hellstrom,and R. Ryhage, J. Lipid Res., 5, 245 (1964). (15) J. Sjovall and R. Vihko, Steroids, 7, 447 (1966). (16) E. C. Homing, C. J. W. Brooks, and W. J. A. VandenHeuvel, “Advances in Lipid Research,” Vol. 6 , Academic Press, New York, N . Y . , 1968, pp 354 -360.
and/or 17~-trimethylsilyloxysteroids exhibited no mass shift in the spectrum of IIc. This indicates a retention of the identity of the TMS groups upon electron impact. Consideration of the spectra of the selectively labeled derivatives IIc and IIIc allows one to identify the ion at mie 143 in the spectrum of IIa and those at mje 143, 169, 182, 198, and 213 in the spectrum of IIIa as containing the 11P-trimethylsilyloxy group. Many of these ions, and particularly rnje 143 and m / e 169, are often characteristic of the presence of an 11trimethylsilyloxy substituent and their mechanisms of formation are presumably similar to those described before (13). The base peak at mje 448 in the spectrum of IIIa is produced by the loss of a methoxyl group from the molecular ion ( m / e 479). The ion at rnje 448 further fragments by the elimination of trimethylsilanol (loss of 90 amu) to give the peak at m/e 358. The spectrum of the selectively labeled analog (IIIc) shows that the latter process involves mostly ( 9 0 z ) the sterically hindered 11/3-trimethylsilyloxygroup, a situation similar to that observed in the spectrum of Ia. By contrast to this the spectrum of the selectively labeled AS-trihydroxy derivative (1Ic) shows that only 50 of the initial electron-impactinduced triniethylsilanol elimination occurs from the 11P ANALYTICAL CHEMISTRY, VOL. 45, NO. 1, JANUARY 1973
9
Table 111. Partial Mass Spectra and Mass Shifts of the Most Significant TMS-ContainingIons in the Mass Spectra of IVa-c IVa(M = 507) IVb IVC [3/9,17a[3@,170(3P-TMS-do; (TMS-ddzI (TMS-de)z] l7a-TMS-de) 156 [51]0 165 165 158 [40] 167 167 170 [26] 179 179 172 [26] 178 178 188 [62] 197 197 364 [25] 373 364 (5)b 373 (1)b 386 [22] 395 386 (3) 395 (1) 476 [lo01 494 485 492 [9] 507 501 507 1341 525 516 Figures in brackets refer to the relative intensities of the ions 01 indicated mass. b Figures in parentheses refer to the ratios of the indicated peaks.
the fourteen mass unit shifts of mje 172 and mje 188 in the spectra of the 20-keto ethyloxime analogs of IVa and Va, it is reasonable to postulate that this series of ions comprises parts of the D ring and the 17p side chain as shown in Scheme 1. Several structures can be written for each of the resulting r - - - - - -1
I
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5
Table IV. Partial Mass Spectra and Mass Shifts of the Most Significant TMS-ContainingIons in the Mass Spectra of VIa-c VIa (M = 450) VIb VIC [38,178[3PJ7P(3P-TMS-do; (TMS-dohl (TMS-ddal 17P-TMS-dg) 73 [32]” 82 73 (1) 82 (3) 130 [17] 139 139 143 [lo01 152 152 255 [4] 255 255 318 [3] 327 318 345 [3] 351 (l)b 345 354 (3)” 360 [7] 369 360 (20) 369 (1) 435 [23] 450 (1) 444 453 (12) 450 [51 468 459 a Figures in brackets refer to the relative intensities of the ions of indicated mass. b Figures in parentheses refer to the ratios of the indicated peaks.
position. It is probable that a conjugated system is produced in the presence of the Ab-olefinic function which enhances elimination from the 3p position in IIa. The mass spectra of the TMS derivatives of both substituted pregnanes IV and V (Table 111, Figure 1) are dominated by the ions produced from the loss of a methoxyl group from the molecular ion to give the peaks at rnje 476 and 474, respectively. The [M-OCH3]+ ion in the spectrum of IVa subsequently eliminates trimethylsilanol from both the 3 p (25 %) and the 17a positions (75%). The effect of the A5 double bond on the specificity of this elimination ( 1 7 ) is again evident in the spectrum of the unsaturated derivative (Va, Figure 1) as the loss of trimethylsilanol from [M-OCH3]+is almost equally distributed between the 30 and 17a positions. In addition to mje 129 in the spectrum of Va, the most characteristic TMScontaining ions in the spectra of IVa and Va occur at mje 156, 158,170,172,and 188 (Table 111, Figure 1). From the spectra of the selectively labeled derivatives IVc and Vc, it was established that, aside from mje 129, these ions contained the D ring TMS group. On the basis of their elemental compositions (determined by high resolution mass spectrometry) and (17) P. Vouros and D. J. Harvey, Clzem. Commun., 1972,765. 10
r--------- !
r--------.
Scheme 1. D ring cleavages leading to characteristic TMScontaining ions in the spectra of the TMS derivatives of 5apregna11e-3p,l7a-diol-20-one methyloxime and 5-pregnene3p,17cu-diol-20-one methyloxime fragment ions, but the present evidence, although defining the groups involved in the formation of these ions, is not sufficient to establish their exact structures. The utility of selective labeling with TMS groups in steroids is further demonstrated by consideration of the spectra of the derivatives of the 17a-methylandrostanes VIa-c-VIIIa-c. The base peak in the spectrum of VIa (Table IV) occurs at m/e 143 and from the spectrum of the selectively labeled derivative (VIc), it is evident that this ion contains the 17ptrimethylsilyl group. 7he mechanism of its formation (Scheme 2) is presumably similar to that proposed by Diekman and Djerassi for the formation of the homologous ion at mje 129 in the mass spectrum of 17~-trimethylsilyloxyandrostane (7). The further decomposition of rnje 143 to the trimethylsiliconium ion of rnje 73 is supported by a metastable peak at mje 44.5. The spectrum of the selectively labeled derivative VIc indicates that the initial elimination of trimethylsilanol from VIa occurs mainly (96%) from the tertiary (17p) position and a similar preference ( 9 0 x ) is apparent in the unsaturated derivatives VIIa and VIIIa. In the spectra of the latter two compounds, the selective introduction of a TMS-d9group in the 17p position shows that the structurally significant TMScontaining ions at rnje 129 and rnje 131 contain the 3p-trimethylsilyloxy group whereas the analogous mje 143 ion in the spectrum of VlIa (mje 157 ion in VIIIa) originates from the D ring function, An additional feature of the mass spectrum of VIIa, which can be readily resolved by the use of
ANALYTICAL CHEMISTRY, VOL. 45, NO. 1, JANUARY 1973
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Scheme 2. Mechanism of formation and fragmentation of the m/e 143 ion in the spectrum of the trimethylsilyl derivative of 17a-methyl-5a-androstane-3P,17P-diol
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Figure 2. Top: Mass spectrum of 3~,17~-bis(trimethylsilyloxy-~~)-l7~-methyl-5-androstene (M = 448) Center : Mass spectrum of 3~,17~-bis(trimethylsilyloxy-d~)-17a-methyl-5-androstene (M = 466) Bottom: Mass spectrum of 3~-~trimethylsilyloxy-~~)-l7~-(trimethylsilyloxy-~~)-l7~-methyl-5-androstene (M = 457) ANALYTICAL CHEMISTRY, VOL. 45, NO. 1, JANUARY 1973
11
Table V. Partial Mass Spectra and Mass Shifts of the Most Significant TMS-ContainingIons in the Mass Spectra of VI1Ia-c VIIIC VIIIa VIIIb [3P,17P(3P-TMS-do; [3P,17B(TMS-ddzI 17P-TMS-dg) (TMS-do)rl 129 [34P 138 129 131 [24] 140 131 157 [loo] 166 166 185 [23] 185 185 211 1261 211 211 226 [20] 226 226 241 [19] 241 241 253 [39] 253 253 267 [59] 267 267 282 [80] 282 282 316 [lo] 325 316 343 [24] 352 343 (1)b 352 (3)* 372 [23] 381 372 (20) 381 (1) 433 [ll] 451 442 447 [3] 462 456 462 [2] 480 471 a Figures in brackets refer to the relative intensities of the ions of indicated mass. b Figures in parentheses refer to the ratios of the indicated peaks.
the selective TMS-d9labeling technique involves the loss of a methyl group from either the M+. or the (M-90)+. ions to give the peaks at rnje 433 and rnje 343, respectively. The spectra of the labeled derivatives of the 17a-methyl (VII6,c; Figure 2,b,c) and 17a-ethyl (VIIIb,c; Table V) compounds provide sufficient isotope or substituent labeling to show that 40x of the methyl loss from M'. originates from the 3/3TMS function and the remaining 60 represents elimination of the 17a-methyl group. In addition the spectrum of the selectively labeled derivative VIIc (Figure 2c) clearly shows that the (M-90)+. ion in the spectrum of VIIa consists of two different species, each eliminating a methyl group with differ-
ent relative preference. This is indicated by the 10 :1 ratio of rnje 352 :rnje 367 as compared to a 1 :4 ratio of rnje 343 :mje 358 (Figure 2c). The (M-90)+. ion originating from elimination of trimethylsilanol from the 38 position, exhibits favorable loss of the 17a-methyl group. By contrast the loss of methyl from the other (M-90)+. ion is much less favorable and is distributed between the 17a and the angular methyl groups. CONCLUSIONS
The data presented above show that it is possible to prepare mixed TMS-do and TMS-dQ steroidal ethers in which the labeled trimethylsilyl moiety occupies a specific position. Unlike previous investigations, which employed an on-column exchange method, we were able to prepare selectively labeled TMS-d9 derivatives in solution by using standard silylation procedures. As illustrated in the examples shown in Figures 1 and 2, the selective silylation in solution, with careful control of the experimental conditions, enabled us to prepare consistently derivatives with an isotopic purity and specificity of 90 or more. This compares with a purity of 40-70 %, obtained by the on-column exchange method, but which usually varied widely with the general condition (e.g., temperature, previous use, etc.) of the gas chromatographic column. As shown in the exatriples discussed here, the preparation of these derivatives is particularly helpful in the interpretation of the mass spectra of trimethylsilyl steroidal ethers. Much of the mass spectrometric information could, otherwise, only have been arrived at by elaborate deuterium and 180-labeling. In view of their simplicity, the selective TMS-dQlabeling procedures described here should be readily applicable to other classes of compounds.
x
RECEIVED for review July 5, 1972. Accepted September 18, 1972. This work was supported by grants from the National Institute of General Medical Sciences (GM-13901, GM16216).
Application of Pattern Separation Techniques to Mass Spectrometric Data Determination of Hydrocarbon Types and the Average Molecular Structure of Gasoline D. D. Tunnicliff and P. A. Wadsworth Shell Development Company, Houston, Texas The mass spectra of a large group of pure compounds typical of those found in gasoline have been used to derive a set of weight vectors which can be used to determine the average properties of gasoline samples. The method is illustrated for the determination of hydrocarbon types and for the determination of the structural features of the average molecule.
structure of a molecule from its mass spectrum. It has also been shown ( 4 ) that data from several different sources can be combined in a single calculation. One of these techniques (3) can also be applied to the determination of molecular types and the structure of the average molecule in a complex mixture. This approach is based on the following set of
ITHAS BEEN DEMONSTRATED (1-3) that pattern separation techniques are powerful tools in the identification of the molecular
(2) P. C . Jurs, B. R. Kowalski, and T. L. Isenhour, ibid. 41, 21 (1969). (3) B. R. Kowalski, P. C . Jurs, and T. L. Isenhour, ibid. p 695. (4) P. C . Jurs, B. R. Kowalski, T. L. Isenhour, and C . N. Reilley, ibid.,p 1949.
(1) L. R. Crawford and J. D. Morrison, ANAL.CHEM.,40, 1469 (1968). 12
ANALYTICAL CHEMISTRY, VOL. 45, NO. 1, JANUARY 1973
