ftiochimica et Biophysics Acta, 296 (1973) 431-4.

45
6 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

BBA 56198

THE USE OF TRIMETHYLSILYL ETHERS IN THE CHARACTERIZATION

OF NATURAL STEROLS AND STEROID DIOLS BY GAS
CHROMATOGRAPHY-MASS SPECTROMETRY

C. J. W. BROOKS, W. HENDERSON and G. STEEL

Chemistry Department, University of Glasgow, Glasgow Gr2 8QQ, (Great Britain) and
Department of Chemistry, university of California, Berkeley, Cali$ 94720 (U.S.A.)
(Received August zSth, 1972)

SUMMARY

Trimethylsilyl ethers of I 6 sterols, six cholestanediols, six cholestenediols and a

cholestanetriol have been examined by combined gas chromatography-mass spectro-
metry. Characteristic features of the data are discussed in relation to previous work,
and examples are given of the use of the technique in the identification of naturally-
occurring steroids.

INTRODUCTION

Trimethylsilyl ethers have been extensively used for the separation and char-
acterization of sterols by gas-liquid chromatography’-’ and combined gas chro-
matography-mass spectrometry 4- l1 . Their extremely non-polar nature is convenient,
for example, in thin-layer chromatography’2*‘3 and aids the selective removal of
hydroxylated materials such as sterols, from the large quantities of extraneous material
frequently encountered in biological and geochemical’4 work. The present study,
initiated in 1967 as an extension of earlier work’, surveys the utility of trimethylsilyl
ethers for the identification of a range of sterols (with 1-3 hydroxyl groups) by gas
chromatography-mass spectrometry. The origin of many of the characteristic frag-
ments in the mass spectra of these compounds is discussed, and the application of the
results to the identification of natural steroids is briefly illustrated.

MATERIALS AND METHODS

Gas chromatography
A Pye 104 gas chromatograph with dual flame ionization detectors was em-
ployed. Chromatography wascarried out using 1.5 m silanised glass columns of o.gj-cm
Abbreviations: the following trivial names of steroids are used: epicholesterol, j-cholesten-3a-
01; ~a-campestanol,(zqi?)-~-methyl-glx-cholestan-3~ol; campesterol, (24R) -z4-methyl-s-cholesten-
3b-01; brassicasterol, (24R)-z4-methyl-~,(E)-22-cholestadien-3&ol; hunisterol, (24R)-24-methyl-9j,
roz-cholesta-5,7,(E)-zz-trien-3&o]; stigmasterol, (zer)-z4ethyl-5,(E)-zz-cholestadien-38-l.
432 C. J. W. BROOKS et al.

internal diameter. The packings used were as follows: I % SE-30 on 100-120 mesh Gas
Chrom Q (Applied Science Laboratories Inc., State College, Pa.); I % OV-17 on 8o-IOO
mesh Chromosorb G-HP (Supelco Inc., Bellefonte, Pa.).

Gas chromatography-mass spectrometry

Most of the tabulated mass spectral data were obtained using an LKB 9000
gas chromatograph-mass spectrometer (LKB-Produkter AB, Stockholm). The elec-
tron energy was 70 eV, the He flow rate from the chromatograph was 25-30 ml/min,
the ion source temperature 270 “C and the molecular separator temperature 250 “C.
The organic geochemical applications were carried out on a Du Pant 21-492-1
high-speed pumping mass spectrometer (Du Pont Instruments, Monrovia, Calif.)
which was coupled directly to a Varian Aerograph 204 chromatograph, without the
use of a separator. The He flow rate from the 9.1 m x 0.076 cm packed glass capillary
columns used in this instrument was 2-5 ml/min and the connecting line was main-
tained at 290 “C. Spectra were recorded at 70 eV. Trimethylsilyl ethers of three sterols
previously examined’ at 20 eV were included in the present study.

Sterols
5a-Cholestane-5,6a-diol was prepared from 5-cholestene”; 5a-cholestane-3/I,
5-diol, 5-cholestene-3/?.7/3-diol and 5-cholestene-3/?,7adiol were prepared from chol-
esterol’6-‘8; 5a-cholestane-3/?,6/&diol and 5a-cholestane-3/3,6a_diol were obtained by
reduction of 3/?-hydroxy-5a-cholestan-6-one with NaBH, and sodium-ethanol16,
respectively. 5a-Campestanol and 5a-ergostanol were prepared by catalytic reduction
of campesterol and brassicasterol, respectively. 26-Hydroxycholesterol was prepared
from kryptogenin.
5-Cholesten-3a-ol (Mann), 5-cholestene-3jI,2oa-diol (Ikapharm) and 5a-chol-
estane-3/3,5,6/%triol (Steraloids) were commercial samples. Other sterols were received
as gifts (see Acknowledgment).

Preparation of trimethylsilyl ether derivatives

For unhindered hydroxyl groups, trimethylsilyl ethers were prepared at room
temperature in pyridine solution using hexamethyldisilazane and trimethylchlorosilane
(IO : I, v/v). After I h, the reagents were removed in a stream of N2 and the residue was
dissolved in ethyl acetate. Insoluble material was removed by centrifugation.
Complete etherification of diols and triols containing hindered hydroxyl
groups was accomplished using a mixture of N-trimethylsilyl imidazole, bis (tri-
methylsilyl) acetamide and trimethylchlorosilane (3: 3:2, v/v/v) at 6o “C for 60 h
(ref. 19) in a “Reacti-Vial” (Pierce Chemical Co.). Reagents were partially removed in
a stream of Nz, water was added and the trimethylsilyl ethers were extracted with ethyl
acetate. The ethyl acetate solution was dried over anhydrous Na,SO, and concentrated
in vacua.

Isolation of puriJed sterol fractions from geological samples

Crude sterol-containing fractions (usually highly-pigmented) were isolated
from sediments by Silica Gel column chromatography of benzene-methanol (3 : I, v/v)
extracts. Trimethylsilyl ethers were prepared as described above. Preparative thin-
layer chromatography of total etherified fractions was carried out on 20 cm x 20 cm
USE OF TRIMETHYLSILYL DERIVATIVES OF STEROLS 433

plates coated with Silica Gel G (E. Merck, Darmstadt; 0.25 mm) using heptane-benze-
ne (I :I, v/v) as mobile phase. An area of Silica Gel representing R, 0.38-0.62 was
scraped from the chromatogram after location by comparison to the mobilities of
reference sterol trimethylsilyl ethers. Most of the pigments and other extraneous
materials remained at the origin in this non-polar solvent system, The purified sterol
trimethylsilyl ethers were eluted from the silica gel by “sonication” in ethyl acetate at
room temperature.

RESULTS AND DISCUSSION

The gas chromatographic retention indices of the trimethylsilyl ethers are given
in Table I. The IO most abundant high mass ions in each mass spectrum (70 eV) are
recorded in Table II together with the relative intensity of the molecular ion. Only ions
above m/e IIO have been tabulated, in order to avoid citation of trivial ions of low

TABLE I
RETENTION DATA FOR STEROL TRIMETHYLSILYL ETHERS
Gas chromatography conditions: 1.5 m I % SE-30 at 230 “C; 1.5 m I % OV-17 at 240 “C

Position I TMS
etherified SE-30 ov-I7 -

5-Cholesten-3a-ol 3a 3005 3140

5-Cholesten-38-01 38 3100 3230
5a-Cholest-7-en-38-01 38 3155 3285
5a-Cholestan-ra-ol 1(x 2910* 3035
5a-Cholestan&-ol 6a 3015 3120
Lumisterol 3B 3010 3175
5,z2-Ergostadien-3&ol 3B 3140 3270
5a-Ergosta-7,22-dien-38-01 38 3190 3410
5a-Ergost-7-en-3-j-01 38 3235 3380
5a-Ergost8(rq)-en-3/I-o1 38 3185 3330
5aCholestane-3/3,5-diol 38 3245 3430
5a-Cholestane-5,6a-diol 6a 3140 3270
5a-Cholestane-5,6/I-diol 68 3115 3275
5-Stigmasten-ga-01 3a 3210 3330
5-Stigmasten-3pol 38 3280 3420
5a-Stigmastan-ja-01 3a 3200 3300
5a-Lanost-7-en-38-01 38 3285 3395
5a-Lunost-g(r &en-3/I-01 38 3265 3375
4-Cholestene-3~,6&liol 3W6B 3165 3220
5-Cholestene-3jI,4jl-diol 38,48 3240 3305
5-Cholestene-38,7a-diol 38,7a 3110 3160
5-Cholestene-38,7/I-diol 38978 3225 3295
5-Cholestene-3&2oa-diol 38,2oa 3295 3340
5-Cholestene-38,26-diol 3W26 3420 3535
5a-Cholestane-3B,6j-diol 38368 3210 3260
5a-Cholestane-3j7,5-diol 3i%sa 3200 3255
5a-Cholestane-5&a-diol 5aAa 3100 3130
5a-Cholestane-3/X5,6/?-trio1 3/X6/3 3325 3430
5a-Cholestane-3/?,5,6/5%-iol 38,5a,68 3220* 3205

* 1.2 m I % SE-30 at 225 “C.

TABLE 11
MASS SPECTROMETRIC DATA FOR STEROL TRIMETHYLSILYL ETHERS (70 eV): THE TEN MOST ABUNDANT PEAKS ABOVE m/e I IO
ARE CITED FOR EACH COMPOUND
Molecular ion is italicized. Peaks reasonably attributableto isotopic composition have been omitted.
“M-l5.B~--I29.C~-_90.D~--105(~,15).E~--I3I.F~-2I9(90,I29). G M-180 (2 x90). ” M--go-side-chain. ’ M-side-chain--H. ’ (CH,),-
Si+OCH-CH=CH2.K M-18

Sterol (trimethylsilylether) Base Molecular Principal ions in mass spectrum

(Mobwt. in parentheses) peak ion % of
base peak

5-Cholesten-ja-ol(458) 129’ 8 121 II9 329’ I45 368= I47 I35 I33 161
(40 %) (37 %) (34 %) (27 %) (25 %) (25 %) (25 %) (24 %) (19%)
5-Cholesten-38-ol(458) 129’ 18 3298 368c II9 121 I45 353D 458 I59 I47
(60 %) (37 %) (31%) (29 %) (25 %) (20 %) (I8 %) (17%) (16%)
5a-Cbolest-7-en-3&ol(458) 255n 92 458 I47 213 I35 II9 I33 229 121 I3I
(92 %) (54 %) (47 %) (39 %) (39 %) (34 %) (31%) (31%) (27 %)
5a-Cholestan-ra-ol(46o) 37o= 18 215 I35 I47 257H 121 I29 I49 161 II9
(58 %J (38 %) (35 %) (33 %) (32 %) (30 %) (29 %) (29 %) (27 %)
5a-Cholestan-6a-ol* (460) 37o= 7 44sA 215 35sD 216 I47 230 I35 2I7 I97
(72 %) (45 %) (32 %) (30 %) (20 %) (19%) (18%) (18%) (16%)
Lumisterol(468) 363D 28 253n 337c I57 378’ I58 I43 211 I59 343
(60 %) (48 %) (39 %) (37 %) (36 %) (35 %) (33 %) (32 %) (29 %)
5,22-Ergostadien-3B-ol(470) 129’ 20 I25 255= I33 II9 I45 380~ I59 I47 121
(91%) (41%) (34 %) (32 %) (29 %) (29 %) (28 %) (26 %) (22 %)
ga-Ergosta-7,22-dien-3j&ol(470) 255n 47 343’ I47 229 470 II9 I33 I45 I35 121
(72 %) (63 %) (61%) (47 %) (46 %) (46 %) (43 %) (40 %) (39 %)
5a-Ergost-7-en-j/?-01(472) 472 100 255n I47 213 II9 I33 229 I35 121 I45
(98 %) (45 %) (42 %) (40 %) (36 %) (33 %) (31%) (30 %) (29 %)
ga-Er.gost-8(I4)-en-3/3-o1(472) 472 100 I47 229 I33 213 II9 I35 25SH 457* I45
(51%) (38 %) (36 %) (33 %) (31%) (31%) (24 %) (24 %) (23 %)
5a-Ergo&an-3Bol* (474) 215 33 216 459* I47 217 369D 474 121 305 230
(58 %) (51%) (45 %) (41%) (35 %) (33 %) (33 %) (32 %) (30 %)
5a-Campestan-38-ol* (474) 216 121 D
215 49 459* 474 217 I47 230 305
(59 %) (55 %) (49 %) (42 %) (41%) (37 %) (33 %) (33 %) ?3?%)
5a-Cholatane-3fi,5-diol(476) I43 I I42 458K 129’ I45 368 I47 II9 121 329
(mono ether) (60 %) (60 %) (35 %) (25 %) (23 %) (23 %) (22 %) (21%) (17%)
5a-Cholestane-5,6c+diol(476) 461~ 23 321 369 I29 368 I35 161 386= I97 476
(mono ether) (76 %) (71%) (48 %) (45 %) (34 %) (32 %) (27 %) (25 %) (23 %)
TABLE II (continued)
_.
Sterol (trimethyIsily1 ether) Base Molecular Principal ions in mass spectrum
(Mol. wt in parentheses) peak ion ‘A of
base peak

5a-Cholestane-5,6&diol* (476) 321 38 461 369 368 476 386 458K 147 353
(mono ether) (79%) (63%) (58%) (38%) (37%) (24 %) (23%) (19%)
5-Stigmasten-38-ol* (486) 129' 22 357B 396c 121 145 215 I47 381~ I59 119
(98%) (66%) (60 %) (46 %) (42%) (41%) (40%) (40%) (38%)
5a-Stigmastan-3a-ol(488) 215 14 216 I47 398’ 217 135 383D 121 I49 230
(46 %) (45%) (36 %) (35 %) (34%) (30 %) (28 %) (27%) (26%)
ga-Lanost-7-en-3jf-o1(5oo) 395D 14 I47 I35 II9 I75 I 29’ I33 121 I45 485*
(70 %) (50%) (39 %) (30 %) (30 %) (29 %) (29 %) (29%) (27%)
5a-Lanost-g(I I)-en-3&01(500) 395D 18 I 29’ 485A 371 I19 500 135 121 I75 I33
(25 %) (23%) 02 %) (20 %) (18%) (I8 %) (17%) (16%) (13%)
5-Cholestene-3&4&diol(546) 366= 35 456’ I47 129’ 327F 417’ 133 119 546 I43
(90 %) (90%) (69%) (49%) (48 %) (48 %) (41%) (35%) (35%)
5-Cholestene-3B,7a-diol(546) 456' 3 129’ 1x9 I45 143 147 159 546 208 233
(8 %) (6%) (6 %) (5 %) (4%) (4%) (3 %) (3%) (3%)
5-Cholestene-3/?,7&diol(546) 456' 3 129’ 119 145 143 I59 546 208 233 I57
(7 %) (5%) (4%) (4%) (4%) (3 %j (3%) (3%) (3%)
g-Cholestene-3&oa-diol(546) 201 I 117 143 461 129’ 281 II9 145 I57 I33
(37 %) (27%) (IS%) (II %) (7 %) (7 %) (7 %) (6%) (5%)
5-Choleatene-3/?,z6-diol(546) 456' 50 129’ 417' 546 441D 121 II9 I45 255 327F
(96 %) (89%) (50 %) (34 %) (29 %) (28 %) (17%) (16%) (15%)
4-Cholestene-3j,6j-diol(546) 403 42 456’ 546 143 441D 194 53I* 147 :2:x, 283
(77 %) (42%) (32 %) (32 %) (28 %) (26 %) (25 %) (16%)
ga-Cholestane-3/I,5-diol(548) 458' I 143 243 142 368o I 29’ I47 215 1x9 117
(70 %) (65%) (27 %) (24 %) (20 %) (20 %) (14%) (12%) (12%)
5a-Cholestane-38,68-diol(548) 458' II I47 368O 369 131 129’ 133 135 228 213
(99 %) (70%) (68 %) (63 %) (62 %) (62 %) (56 %) (54%) (47%)
5a-Cholestane-5,6a-diol(548) G 129’ 184
533A 42 321 369 147 548 183
(79 %) f5Y%j (49 %) (47 %) (42 %) (38 %) (38 %) (27%) ;:%I
5a-Chole&ane-3jl,5,6&triol 129’ 1 403 321 456 I43 546K 147 119 459 367
(his ether) (564) (96 %) (84%) (76 %) (45 %) (45 %) (40 %) (40 %) (29%) (22%)
5a-Cholestane-38,5,6/Miol 456O 4 546’ 321 129’ 403 217 367 147 230 265
(tris ether) (636) (62%I (58%) (57%) (51%) (44%) (38 %) (32 %) (25 %) (19%)
* Spectra run on Du Pent 21-492-I gas chromatography-mass spectrometry system at 70 ev.
436 C. J. W. BROOKS et al.

mass. Where ions of lower mass or of low abundance have been deemed to be of
diagnostic importance, these are discussed separately in the text.

ga-Cholestan-la-01
The base peak in the spectrum of the trimethylsilyl ether of this compound is
at m/e 370 (M-go).* This ion has also been found to be extremely abundant in the
spectra of trimethylsilyl ethers of 5a-cholestan-2/I-, 3a- and 3/L01’ and ga-cholestan-
6a-ol (discussed below). Also important in the mass spectra of these cholestanol
trimethylsilyl ethers are m/e 215 and 216, the latter always more intense than can be
accounted for by the normal isotope effect from m/e 215. Although not recorded in
Table II, m/e 315 appears to be characteristic of the C-I trimethylsilyloxy group and
could possibly result from the process shown in Scheme I, involving loss of Ring A
together with the trimethylsilyloxy group.

ga-Cholesran-6a-ol
The mass spectrum of the trimethylsilyl ether is somewhat similar to that of the
ra-isomer : however, it lacks m/e 3 15 but shows m/e 305 (M- 155) presumably due to
loss of the side-chain together with 42 mass units from Ring D. Although it is not
among the ten most abundant ions, we have found m/e 321 to be especially char-
acteristic of cholestanes containing the 6-trimethylsilyloxy group. The possible struc-
ture of this ion will be discussed later.

ja-Campestan-j/Sol and ga-ergostan-#-ol [ (zqR)-zq-methyl-ga-cholestan-~~-ol and

(zqS)-zq-methyl-ja-cholestan-3fl-ol)]
Both of these compounds gave the expected stanol trimethylsilyl ether frag-
ments (e.g. m/e 215, 216, 217, 230 and 305)‘~” with no pronounced differences either
in ions produced or in abundances (see Table II). The molecular ion (m/e 474) is a
little more abundant in the case of campestanol, which also has a more intense m/e
306 ion relative to m/e 305 (due to loss of the side-chain and 42 mass units from Ring
D) than does ergostanol trimethylsilyl ether. Examination of these stanols has been
necessary in connection with organic geochemical studies of the steroid content of
recent sediments3’. In sediments, geological reduction of phytosterols, such as brassi-
caster01 and campesterol. results in the formation of 5a-ergostanol and ga-campesta-
nol. Fig. I shows a mass spectrum of a compound which was isolated from a sedi-
ment approximately IOOOOO years old and identified from gas chromatographic and
mass spectrometric data as 5a-ergostanol and/or 5a-campestanol trimethylsilyl ether
slightly contaminated with other material. We are at present unable to distinguish
between the 24-epimers chromatographically.

* Charge symbols are omitted for simplicity.

USE OF TRIMETHYLSILYL DERIVATIVES OF STEROLS

Fig. I. Line diagram of a mass spectrum of a sterol (as trimethylsilyl ether derivative) derived from a
IOOOOO year old sediment. The compound has been identified as ga-ergostanol or Sa-campestanol.

5-Cholestenga-ol and pzholesten-_$I-ol

The mass spectra of the trimethylsilyl ethers of these epimeric sterols are
closely similar (Fig. 2). The mass spectrum of cholesterol trimethylsilyl ether is
dominated by fragments of m/e 129 and m/e 329 (M- 129), the genesis of which has
already been extensively discussed 22,23.7.There are small intensity differences between
the spectra of the 3a- and 3/%derivatives, but these were not accentuated by recording
the spectra at low voltage (15 eV). The isomeric sterols can, however, be very clearly
differentiated by both gas-liquid chromatography (Fig. 2) and thin-layer chromato-
graphy.

ga-Cholest-pen-#ol
The spectrum of the trimethylsilyl ether of this steroid was found to be in good
agreement with previously recorded data6-s. The base peak of the spectrum (m/e 255)
is due to the elimination of the side chain plus trimethylsilanol (I 13 phs 90 mass units)
from the molecular ion. The ion at m/e 229 in the tabulation corresponds to loss of
the side chain (CsHi,) phs trimethylsilanol and an additional. 26 mass units of un-
known origin (probably C-17 and C-16). Also of structural importance, but not of
great enough abundance to be recorded in Table II, are: m/e 345 (6%) which corre-
sponds to loss of the side-chain; m/e 303 (4x), loss of side-chain plus 42 mass units
from Ring D; and m/e 318, loss of side-chain phs 27 mass units. These last three ions
have also been observed from the trimethylsilyl ether of 5a-ergost-7-en-3P-01.

Fig. z. Gas chromatogram (indicating complete separation) of trimethylsilyl (TMS) ethers of chol-
esterol and epicholesterol, and line diagrams of the closely similar mass spectra.
438 C. J. W. BROOKS et al.

ga-Ergostq-en-J/?-o1
The trimethylsilyl ether mass spectrum showed an abundant molecular ion
(m/e 472) and characteristic fragments of m/e 255, 213 and 229. The recognition of this
fragmentation pattern, together with correlation of gas chromatographic retention
times, has permitted the identification of 5cr-ergost-7-en-3/3-ol in a lacustrine algal
sample. Fig. 3 shows the gas chromatogram of the sterol fraction from this alga and
Fig. 4a shows a line diagram of the trimethylsilyl ether of 5a-ergost-7-en-3/-?-ol
obtained by gas chromatography-mass spectrometry of this sterol fraction. Fig. 4b
shows a mass spectrum of a sterol trimethylsilyl ether of longer retention time which
is also clearly a A’-compound but has a molecular ion 14 mass units higher. The re-
tention index difference between the trimethylsilyl ethers of 5-cholesten-3/3-ol and
5a-cholest-7-en-3/?-ol(55 units on I % SE-30 at 230 “C) was the same as that between
the trimethylsilyl ethers of (24R)-24-ethylcholest-5-en-3fl-ol(j3-sitosterol) and the un-
identified sterol: thus, the latter was probably a 24-ethyl-5a-cholest+en-38-01. Both
5a-ergost-7-en-3/Lol and a 24-ethyl-5a-cholest+en-3fi-o1 have previously been iden-
tified in the green alga Oocystis polymorphaz4.

ja-Ergost-8(rq)-en-g/&o1
It has been previously noted’ that the trimethylsilyl ethers of A’- and A8(14)-
cholestenols, though easily distinguished by gas-liquid chromatography, give similar
mass spectra, and we have found corresponding results for A’- and Aso4)-ergostenols.
However, some minor ion abundance differences can be observed : the ions at m/e 255
(M-go- side-chain) and m/e 213 (M-go- side-chain-42) are more abundant in
the A’-sterol derivatives than in their Aeo4)-isomers.

ga-Lanost-9( I I) -ew#ol
As expected from experience with other lanostenol trimethylsilyl ethers, the
base peak of the mass spectrum of the trimethylsilyl ether of this compound corre-
sponds to elimination of 105 mass units (trimethylsilanol plus methyl) from the mole-
cular ion. Of greater interest was the fact that this compound gave a prominent ion at
m/e 371 (M- I 29) and a corresponding ion at m/e 129. Such ions are normally char-
acteristic of A5-3-trimethylsilyl ethers: it seems likely that they arise from the A9-3-
trimethylsilyl ether by the mechanism indicated (Scheme 2). From this information it
seems probable that a compound extracted from human tissue by Miettinen and

Scheme 2
USE OF TRIMETHYLSILYL DERIVATIVES OF STEROLS 439

I
0 4 8 12 16 20
minutes

Fig. 3. Gas chromatogram of a trimethylsilylated sterol fraction obtained from a lacustrine alga. The
chromatogram was obtained on a 3 % JXR column (3 m x 0.32 cm) at 265 “C and the sterols identified
are as follows: (I) cholesterol, (2) brassicasterol, (3) campesterol, (4) stigmasterol, (5) ja-ergost-7-en-
38-01, (6) unidentified, (7) 24-ethylcholest-7-en-38-01.

(0)

(b)

Fii. 4. Line diagrams of mass spectra obtained by comb&d gas chromatography-mass spectro-
metry of the trimethylsilylated algal sterol fraction, the chromatogram of which is shown in Fig. 3.
The’ spectra are of: (a) Peak 5, ja-ergost-7-en-3&ol trimethylsilyl ether and (b) Peak 7,24-ethyl-ga-
cholest-7-en-38-01 trimethylsilyl ether.
440 C. J. W. BROOKS et al.

Luukkainen25 which had a molecular weight of 5oo as the trimethylsilyl ether, and
gave m/e 129 and m/e 371 (M- 129). with the base peak at m/e 395, was a A9(“)-
lanostenol rather than a A5-lanostenol. Eneroth et al.” interpreted a similar mass
spectrum, from a sterol of human meconium, as indicative of 5x-lanost-9(r r)-en-
3fl-01 trimethylsilyl ether.

The fragmentation of the trimethylsilyl ether supports the assignment of this

structure (brassicasterol) to a sterol extracted from human feces” and from lake
mudr4. Prominent in the mass spectrum is an ion of m/e 125 representing the un-
saturated (&Hi,) side-chain. The most abundant ion, not represented in Table II,
is m/e 69, ascribed to cleavage adjacent to the A22 double bond. This ion also forms
the true base peak of 5a-ergosta-7,22-dien-38-01 trimethylsilyl ether.

In addition to abundant ions at m/e 255 and 229, characteristic of A’-sterol

trimethylsilyl ethers, the mass spectrum of the trimethylsilyl ether contains an ion of
m/e 343 due to the loss of the side-chain plus two H atoms, a mode of fragmentation
found in many sterols with side-chain unsaturation’*“s. Orcutt and Richardson24 have
assigned the structure 5a-ergosta-7,22-dien-3fl-ol to a compound isolated from a green
alga. The base peak they recorded for the spectrum of the trimethylsilyl ether at m/e
400 was anomalous: Dr Orcutt has kindly informed us that it appeared to arise from
an unknown contaminant, and should be disregarded.

Lumisterol (gfl,Ioa-ergosterol)
The spectrum of lumisterol trimethylsilyl ether is similar to that of ergosterol
trimethylsilyl ether. Apart from the base peak (M- 105) ions characteristic of a A’,’
sterol trimethylsilyl ether occur at m/e 337 (M- 131) and at m/e 131 (ref. 7). This mode
of fragmentation involves loss of C-I, C-2 and C-3 plus the trimethylsilyloxy group
and an additional H atom, from the molecular ion’. Another characteristic ion occurs
at m/e 343 (M- 125) due to loss of the unsaturated (C9H1,) side-chain.

Trimethylsilyl ethers of dihydroxy and trihydroxy steroids

The incidence of the rearrangement ion of m/e 147 in the mass spectra of
steroid diol trimethylsilyl ethers has recently been discussed26. It may be noted that
ions of this nominal mass are prominent in the spectra of many sterol trimethylsilyl
ethers (cJ Table II and ref. 7).
q-Cholestene-@,6/3-diol. The A4-3/Gtrimethylsilyl ether group gives rise to ions
of m/e 142 and 143 as expected 27. However, these are less abundant than in the mass
spectrum of the trimethylsilyl ether of 4-cholesten-38-01 (ref. 27). The 6-trimethylsilyl-
oxy group evidently directs a competitive fragmentation affording the base peak of
m/e 403, possibly according to Scheme 3.
5-Cholestene-3/I,&diol. The two strongest peaks in the spectrum of the bis
(trimethylsilyl) ether result from losses of trimethylsilanol. It is noteworthy that the
ions at m/e 129 and (M- 129), characteristic of A5-3-trimethylsilyl ethers, are of
comparable intensity. Similar enhancement of the peak at (M- 129) has been noted
for 4-alkylated analogues22,23*7.
USE OF TRIMETHYLSILYL DERIVATIVESOF STEROLS 441

scheme3

M;.Y.lf-s*- p

?OSit$ +OSiMc +OSiMe

3 3
m/e 403

5-Cholestene-$?,7cr-dial and 5-choZestene-3B,yfi-diol. Loss of trimethylsilanol

(M-go) due to facile elimination of the allylic trimethylsilyloxy group at C-7 forms
the base peak in the spectra of the bis(trimethylsily1) ethers of these compounds. There
are no other fragments (other than m/e 73) amounting to more than IO % of the in-
tensity of the base peak.
5-Cholestene-3&zoa-dial. The mass spectrum of the bis(trimethylsily1) ether is
dominated by the Cleavages A and B, a to the trimethylsilyloxy group (Scheme 4).
Cleavage A, between C-20 and C-21, yields the ion of m/e 461 (M-85). Cleavage B
between C-17 and C-20 gives the base peak of the spectrum at m/e 201, corresponding
to the complete side-chain including the trimethylsilyloxy group.

Scheme 4

5-Cholestene-3b,.SdioZ. The mass spectrum of the bis(trimethylsily1) ether does

not show any fragments characteristic of the primary trimethylsilyloxy group at C-26.
A characteristic feature of this compound is its exceptionally long retention time
(Table I). We have not found any d5-cholestenediol trimethylsilyl ether with a longer
retention time. Fragments of m/e 129 and m/e 417 (M- 129) are prominent. Losses of
go mass units and go plus 15 mass units are also clearly visible. Less abundant, but
none the less important, is m/e 327 (M-219) due to loss of 129 mass units plus
trimethylsilanol from the molecular ion. 5-Cholestene-3&26-diol, both in the free
form and as mono- and diesters, has been found to be a characteristic component of
human atherosclerotic aorta1 tissue, where it has been identified by gas chromatography
and mass spectrometry 28*29(Fig. 5). This diol has been found also as a sulphate in
infant feces.”
ga-Cholestane-$&diof. Both the mono- and bis(trimethylsily1) ethers of this
compound contain in their mass spectra abundant ions at m/e 143 and 142 which are
characteristic of the d4-3-trimethylsilyl ether structure. This suggests that during
electron impact the fragmentation of these derivatives may involve elimination of the
5a-functional group together with a H atom from C-4 resulting in the formation of
a A4 double bond. An abundant fragment occurs at m/e 243 in the mass spectrum of
the bis(trimethylsily1) ether: study of the perdeuteriotrimethylsilyl ether30 has shown
that this ion contains two silyl groups, and a possible fragmentation mode is outlined
in Scheme 5.
442 C. J. W. BROOKS et al.

0 ‘ s 12 min 16 20 2.t.

Fig. 5. A total ion current chromatogram of sterols (as trimethylsilyl derivatives) obtained by alkaline
hydrolysis of a human arterial ester fraction. Gas-liquid chromatography was carried out on a 1.8 m,
I % OV-I column at 240 “C and the sterols shown are as follows: A, 5-cholestene-3/?,7a-diol bis-
(trimethylsilyl) ether; B, 5-cholestene-3&7,!-diol bis(trimethylsily1) ether and; C, 5-cholestene-3j?,26-
diol bis(trimethylsily1) ether.

p-Cholestane-5,6u-diol andga-cholestane-3j?,5,6&triol. As previously described,

the gc+hydroxyl group does not form a trimethylsilyl ether under the normal tri-
methylsilylation conditions. Complete etherification can, however, be accomplishedig.
The mono- and bis(trimethylsily1) ethers of the ga,6a-diol and the bis- and tris-
(trimethylsilyl) ethers of the 3/3,5a,6/Striol have been examined by gas chromatography
and combined gas chromatography-mass spectrometry. All these derivatives show an
abundant ion at m/e 321 in their mass spectra. A shift of + 9 mass units in this ion on
formation of the perdeuteriotrimethylsilyl ethers 3o has shown that in each case the
fragment contains only one trimethylsilyl group. Examination of the mass spectrum of
the bis(trimethylsily1) ether of ga-androstane-3&5,6a-trio1 has shown absence of the
ion at m/e 32 I and the appearance of an ion of m/e 209 (- I I 2 mass units) indicating
that the fragment of m/e 321 contains the CsH,, side-chain. A comparison of the two
USE OF TRIMETHYLSILYL DERIVATIVES OF STEROLS 443

Fig. 6. Line diagrams of the mass spectra of (a) 5a-androstane3/?,5,6cc_triol bis(trimethylsily1) ether
and (b) ga-choleatane3/&6~-trio1 bis(trimethylsily1) ether.

spectra is shown in Fig. 6. By analogy with the fragmentation of 5-hydroxy-d

dimethylamino-ga-cholestanes’r it is proposed that the ion of m/e 321 arises from the
process shown in Scheme 6. An ion at m/e 403 observed from all these cholestane
derivatives, and shown to contain the side-chain and one trimethylsilyloxy group,
probably results from loss of Ring A.
We have already used data for the trimethylsilyl ethers of 5a-cholestane-5,6a-
diol in an investigation of the structure of a cholestene isolated from atherosclerotic
human arteries3*. The arterial cholestene was treated with 0~0, and the resulting diol

Scheme 6

Tj$(%$&”

tOSiM~3 +OSiMe
3

m/e 321
444 C. J. W. BROOKS et al.

was trimethylsilylated. Gas chromatography-mass spectrometry showed that only a

mono(trimethylsily1) ether had been formed. An ion at m/e 321 suggested that one of
the vicinal hydroxyl groups was at C-6 in the steroid nucleus. Since a 6+dihydroxy
compound would not have had a strongly hindered hydroxyl group, it was inferred
that the hydroxyls introduced by the action of 0~0, were at C-5 and C-6, i.e. of 5-
cholestene.
The results surveyed above substantially extend the available reference data
for gas chromatography-mass spectrometry of sterol trimethylsilyl ethers. The mass
spectrometric fragmentations of these derivatives, when interpreted in conjunction
with gas chromatographic retention values determined on two different liquid phases,
provide powerful evidence for identification. The present methods do not distinguish
between epimeric 2,+methylsterols, but apart from this limitation gas chromatography-
mass spectrometry is by far the most effective technique for characterising structural
and stereochemical features of sterols in microgram quantities of samples. Trimethyl-
silyl ethers are generally the derivatives of choice for initial studies: where additional
character&&ion of particular sterols is necessary, a wide range of other derivatives is
available, and in some instances gas chromatography-mass spectrometry of the free
sterols may be highly definitive.

ACKNOWLEDGMENTS

The authors are grateful to: Dr P. Bladon for samples of got-ergost+en-3fl-01,

5a-ergost-8(14)-en-38-01, 5a-ergosta-7,22-dien-3/I-o1 and lumisterol; Dr G. Eglinton
for 5a-cholestan-6a-01; Prof. Sir Derek Barton, F.R.S. for 5a-lanost-pen-3P-ol and
5a-lanost-g(II)-en3fi-01; Prof. H. B. Henbest for ga-cholestan-Ia-01; Prof. R. P.
Cook for 5-cholestene-3/3.&I-diol and +cholestene-3/?,6/3-diol; Dr G. F. Woods for
24-ketocholesterol; Dr W. McCrae for kryptogenin. Thanks are due to Miss G. M.
Sharples and Mr J. D. Gilbert for their helpful contributions. One of us (C.J.W.B.) is
also indebted to Prof. E. C. Horning for his encouragement.
This work was supported by grants from the Medical Research Council,
N.S.F. (GB-2ggo2X) and N.A.S.A. (NGL-og-oo3-003). The LKB gooo gas chro-
matograph-mass spectrometer (Glasgow) was provided by SRC grant No.B/SR/23g8.

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