Organic Mass Spectrometry, 1973, Vol. 7, pp. 925 to 948. Heyden & Son Limited. Printed in Northern Ireland

MASS SPECTRA OF TRIMETHYLSILYL ETHERS OF SOME A5-3j3-HYDROXY C 1 g STEROIDS

C. J. W. BROOKS Chemistry Department, The University, Glasgow G12 SQQ, Scotland

D. J. HARVEY, B. S. MIDDLEDITCH and P. Vou~os Institute for Lipid Research, Baylor College of Medicine, Houston, Texas

77025, USA.

(Received 9 October 1972; accepted (reaised) 8 January 1973)

Abstract-The mass spectra (20 eV electron energy) of a wide range of A5-3B-hydroxy C,, steroid TMS ethers have been examined with the aid of high-resolution mass measurements, together with deuterium and oxygen-18 labelling data. The validity of many previously proposed fragmentation modes has been confirmed. A number of ions regarded as diagnostic have been shown to be less specific than had been formerly supposed. Several novel fragmentations have been observed and investigated.

I N T R O D U C T I O N

CHOLESTEROL is present in practically all living organisms, and is the primary source of mammalian steroidal hormones. It may, for example, be metabolised to pro- gesterone via 20,22-dihydroxycholesterol and pregnenolone. Alternatively, complete oxidative removal of the side chain leads to dehydroepiandrosterone (I) and to the C,, hormonal steroids. The A5-3j3-hydroxy steroids are precursors of hormones with the A4-3-one structure.l Consequently, A5-3p-hydroxy C,,-steroids (or their con- jugates) are found in quantity only if 3,%hydroxysteroid dehydrogenase is not actively converting them to A4-3-keto steroids. This situation is encountered, for example, in newborn mammals and in certain pathological conditions.2

The technique of combined gas chromatography-mass spectrometry (g.c.-m.s.) has been used in the identification of a large number of A5-3j3-hydroxy C,,-steroids, as their trimethylsilyl (TMS) ether derivatives. The following are representative of many samples studied: urine3 to and f a e c e ~ ~ ~ ~ . ~ of newborn and infant humans, meconium of newborn humans,7 human umbilical cord p l a ~ m a , ~ human amniotic fluid,1° human bile,,, human peripheral plasma,12 to l5 plasma and urine of an eight- year old boy with 3p-hydroxy steroid dehydrogenase deficiency,16 urine of a pregnant irus monkey17 and of a newborn chimpanzee,18 and urine and faeces of female germ- free and ‘conventional’ rats treated with a 3~-hydroxy-A5-oxidoreductase inhibitor.lg G.c.-m.s. has also been used to show that A5-3/3-hydroxy steroids were not excreted by an anencephalic newborn infant.20

Despite the clinical significance of androstenols,21 there has been no systematic survey of the mass spectral fragmentations of their derived TMS ethers. We have accordingly examined the low-resolution mass spectra of a number of these compounds, and with the aid of isotope-labelling experiments and high-resolution mass measure- ments, have elucidated some routes of fragmentation.

925

926 C. J. W. BROOKS, D. J. HARVEY, B. S. MIDDLEDITCH and P. VOUROS

RESULTS AND DISCUSSION

The use of an electron energy of 70 eV in the measurement of mass spectra of many compounds gives a multiplicity of abundant fragment ions of relatively low mass. Often, these are of limited structural significance: few of the informative ions in the mass spectra of steroids and their derivatives have mje values less than 100. Spectra obtained using lower electron energies generally contain fewer ions and these, comprising mainly products of primary, secondary or tertiary fragmentations, often afford a clearer insight into the processes taking place in the ion source. Spectra obtained at 20 eV have therefore been used in this study.

Mass spectra of TMS ethers of representative androstenols (I to XIII) are shown in Figs. 1 to 13. Tabulated data for these, and for other spectra referred to in the

TMSO TMSOe

0 ,111

& QTMS MS

TMSO (VIII)

Mass spectra of trimethylsilyl ethers of some A5-3P-hydroxy C,, steroids 927

TMSO (XIII)

text, have been submitted to the Mass Spectrometry Data Centre (Aldermaston, Berkshire, England).

Fragmentations of androst-5-en-3p-ol TMS ether

The spectrum of androst-5-en-38-01 TMS ether (11, Fig. 2), the simplest compound of the series, contains many ions which are characteristic of TMS ethers of A5-3p- hydroxy C,,-steroids and analogous compounds with side-chains at C-1 7.22 The investigations of Diekman and DjerassiZ3 on labelling of cholesterol with deuterium or other substituents were particularly useful in assigning origins to many of the common ions (Scheme 1):

[M - 90]+. (a): elimination of the 3p-trimethylsilyloxy moiety with a hydrogen atom, mainly from C-4 ( p ) ;

[M - 105]+ (b) : loss of a methyl radical from a. A metastable ion attests to this fragmentation in the spectrum of I1 and the methyl group probably originates from c-19;

[M - 15]+ (c): by analogy with cholesterol TMS ether, it is likely that this ion is formed by loss of a methyl radical from the TMS group, there being no metastable ion for loss of trimethylsilanol from c;

2

928 C. J. W: BROOKS, D. J. HARVEY, B. S. MIDDLEDITCH and P. VOUROS

nije 346 (17%)

(e ) ni/e 217 (100%). I

(a) rn/c 256 (45 %) (k) in/e 213 (2 %)

(l)ni/e 135 (37%) (i) ni/e 214

SCHEME 1

[M - 129]+ ( e ) and m/e 129 (d): these ions are formed by scission of C-1/10 and C-3/4, with hydrogen transfer from C-2 to C-4. The genesis of these ions has been fully inve~tigated.~~ It should be noted, that whereas A5-3,8-hydroxy steroid TMS ethers give rise to ions at m/e 129, A4-3/3-hydroxy steroid TMS ethers afford characteristic ions at nile 142 and 143.24

Mass spectra of trimethylsilyl ethers of some A5-3P-hydroxy CI9 steroids

I ' I I 0

m r!

0 m 0

0 m

0

0 0 N

=t

m 0 Y- 0 o

0 v,

f W

929

c i . 9

r- N

0 9

930 C. J. W. BROOKS, D. J. HARVEY, B. S. MIDDLEDITCH and P. Vou~os

Ions of m/e 73 (f) and 75 (8) appear in the spectra of almost all TMS ethers, but are of little diagnostic significance. They are of relatively low abundance in most 20 eV spectra.

Ring D of the steroid nucleus may undergo fragmentation by several r n ~ d e s . ~ ~ . ~ ~ There is no appreciable degree of such fragmentation of 11, but the [M - 90]+. ion (a) undergoes typical ring D fragmentations. Elimination of (2-16 and C-17, with substituents, leads to an ion (12) of m/e 228. Loss of C-15 to (2-17 gives an ion (i) of m/e 214. It has been postulated that ions of this type may further eliminate a methyl radical to afford an ion ( j ) of nzje 199.23 Loss of an additional hydrogen atom (from C-8 in some analogous compounds which have been studied by deuterium labelling) with C-15 to C-17 produces a 'nuclear' fragment ion (k) of m/e 213. This ion is of moderate intensity in the spectra of compounds with substituents in ring D and for such compounds, is the result of the favoured fragmentation mode of this ring.

The abundant ion of m/e 135 (1) is probably formed in the same way as that in the spectrum of 5a-androstane: it would comprise rings C and D, from which a hydrogen atom has been lost.25

A further significant ion appears at 7n/e 121 (nz). This ion is present in the majority of the spectra of I to XI11 and also in many previously reported spectra of compounds of this type, but its significance has been obscured by the presence of other ions of similar mass, particularly in the 70 eV spectra. Moreover, an ion of m/e 121 is observed in the spectrum of cholesterol TMS ether, and is shifted to m/e 135 and 149 in the spectra of 4%-methyl and 4,4-dimethylcholesterol TMS ether, respectively.22 A possible mode of formation of these ions is shown in Scheme 2.

(i) m/e 214 1 1 1

H

(4 in/e 121

SCHEME 2

Mass spectra of trimethylsilyl ethers of some A5-3P-hydroxy C1, steroids 93 1

It should be emphasised that we have no direct evidence for the compositions and structures of the ions shown in Schemes 1 and 2. Their constitutions are inferred by analogy with similar ions in more fully investigated spectra. Nevertheless, such representations provide a useful foundation for investigation of influences of sub- stituents on the fragmentations of other androstenol TMS ethers.

Fragmentations of compounds with keto groups in ring D

An impressive feature of the mass spectrum of 3/l-hydroxyandrost-S-en-l 7-one TMS ether (Fig. 1) is its s i m p l i ~ i t y . ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ The base peak at m/e 129 ( d ) accounts for 38 % of the total ion current: the ketonic neutral fragment appears to accom- modate a large portion of the energy content of the molecular ion. The spectrum of 3/l-hydroxyandrost-5-en-l6-one TMS ether (XIV) is similar to that of the isomeric 17-keto steroid, the ion at [M - 56]+. (m/e 304) being somewhat more abundant in the former. An additional ketone function in ring C, as in 3P-hydroxyandrost-5- ene-11,17-dione TMS ether (111, Fig. 3) , appears to afford increased stability to both the neutral particles and the ions produced by certain fragmentations. The base peak, also at m/e 129, accounts for 61 % of the total ion current.

The identities of ions of types a to e in the spectra of I, I11 and XIV were verified by examination of spectra of the corresponding d9-TMS ethers. In each case, it was found that more than 90 of the [M - 15]+ ions were formed by loss of a methyl radical from the TMS group.

Djerassi and co-workers found that 5a-androstan-17-one and its 16,16-d2 analogue gave an [M - 56]+. ion at m/e 218, while indicating that this ion was formed by fragmentation of ring D, as for ion (i).30 The mass spectrum of compound I contains an ion of type [M - 56]+. at m/e 304, which at first sight appeared to represent a similar fragmentation. Contrary to this view was our finding that the 16,16-d2 analogue of I also contained an ion of type EM - 56]+-. Moreover the spectra of a large number of isotope-labelled analogues of I, I11 and XIV contained ions of type [M - 56]+., but no corresponding ions formed by loss of hydrogen atoms from C-9,

932 C. J. W. BROOKS, D. J. HARVEY, B. S. MIDDLEDITCH and P. VOUROS

n 2

0 0 3 cy I

O?.$

s: I I-

1 - 3

m

m

; ul 0 0 I

Mass spectra of trimethylsilyl ethers of some A5-3P-hydroxy C,, steroids 93 3

C-12, C-15, C-16, C-17 or from the TMS groups of these compounds. By means of l80 labelling, the 17-keto group was found to remain in the [M - 56]+. ion. 6- Methyl- and 4,4-dimethyl-3~-hydroxyandrost-5-en-17-one TMS ethers both gave rise to [M - 56]+- ions. The saturated analogue of I, 3@-hydroxy-5a-androstan-l7-one TMS ether and its 16,16-d2 analogue underwent the expected ring D fragmentation, however, to yield ions of type [M - 56]+. and [M - %I+-, respectively. These data, summarised in Table 1, indicated that the apparently anomalous [M - 56]+. ions (0) were formed by fragmentation of ring A: high resolution mass measurements showed that the neutral particle lost in the formation of such ions had the composition C,K,O. The postulated mechanism31 involves an intermediate rearrangement ion (n) with a TMS group at C-6.

On the other hand, it was found that all of the 56 a.m.u. lost from the [M - 90]+. ion of I in the production of the ion of m/e 214 (i) originated from ring D. Similarly, the ion of m/e 213 (k ) was formed by fragmentation of ring D of the [M - go]+. ion.

( n ) TMS I 4

T M S (0) [M - 561’

TABLE 1. SALIENT MASS SPECTROMETRIC DATA FOR CHARACTERISATION OF THE [M - 56]+’ PEAK IN THE SPECTRA OF I, 111, XIV AND LABELLED ANALOGUES

m/e 304 305 306 307 308 313 318 321 322 323 327 332

100% - ( 0

16,16-d, (82%)” dg-TMS -

17-180 (41 %jb 100 6-Me - 4,4-diMe -

(111) 15,l 5,17,17-d4 (74 %)” dg-TMS -

(XIV) -

170 %)b - dg-TMS -

1008 -

9,12,12,16,16-d, 6 37 - _ 1008

- -

1ooa

a [M - 56]+* Yields of labelled compounds calculated from molecular ions with corrections for 13C (N.B. Values

in Table are relative abundances of multiplets, not corrected for 13Cj.

934 C. J. W. EROOKS, D. J. HARVEY, B. S. MIDDLEDITCH and P. V o u ~ o s

!

The fragmentations outlined in Scheme 3 account for all ions in the spectrum of 111 which have relative abundance greater than 2 % of the base peak.

I [M - 56]+’

m/e 318 (15 %) i [M - 901’’ m/e 284 (1 %)

[M - 105]+ mje 269 (1 %)

[M - 711‘ m/e 303 ( 3 %)

m/e 359 (2 %)

SCHEME 3

The spectrum of 3p,1 I~-dihydroxyandrost-5-en-17-one mOnO(3) TMS ether (IV, Fig. 4) is similar to that of I, but with additional ions corresponding to elimina- tion of the elements of water from ions of type a to c and e. An [M - 56]+* ion was observed at m/e 320, but there was no ion corresponding to an additional loss of water.

Fragmentations of compounds with hydroxyl groups in ring D The presence of a trimethylsilyloxy group in ring D does not afford stability to the

molecular ion, or to major fragment ions to the same extent as a ketone group. This results in the formation of a wider variety of ions, as in the spectra of androst-5- ene-3p,17a-diol TMS ether (V, Fig. 5),I2*l5 and its 178 (XV),12 16a (XVI) and 16p (XVII) isomers. The spectra of XV to XVII are all very similar to that of V, but extensive labelling studies have revealed distinct differences in their fragmentation modes.

I t is interesting to note that there is no ion of type [M - 56]+. in the spectra of these compounds. Abundant fragment ions are produced by sequential eliminations of trimethylsilanol from the molecular ions. 180-Labelling has indicated that initial

Mass spectra of trimethylsilyl ethers of some A5-3&hydroxy CIS steroids

I I I I 0

m

BB 0 (I) I

+-

.*

U m v

3 P E

m2-== m 1 ; a N

i

(I)" 11

% 0 I- v) I

m

Lwt I

D D . al

E

0 0 N

m x

935

936 C. J. W. BROOKS, D. J. HARVEY, B. S. MIDDLEDITCH and P. VOUROS

loss of trimethylsilanol is predominantly from the 3-position: 99% in the case of XVI132*33 and 91 % for XV.33

It has been suggestedz3 and we have recently confirmed by deuterium labelling,34 that ions in the spectra of 17-trimethylsilyloxy steroids at m/e 129 comprise C-15 to C-17 with substituents, less one of the hydrogen atoms at C-16. When there is more than one TMS group in a molecule, the possibility exists that the ions of m/e 129 do not contain the same TMS group. In fact it has been found, that whereas the majority of such ions in the spectrum of 5a-androstane-3a,l7P-diol TMS ether originate from ring D, 15% are formed by fragmentation of ring A. Nevertheless, the presence of a A5 bond strongly directs the fragmentation of ring A: ISO and deu- terium labelling results indicate that the ion of m/e 129 in the spectrum of XVII originates almost solely from this ring.

The origin of an abundant ion at m/e 215 in the spectra of V, XV, XVI and XVII may be formally ascribed to sequential loss of a fragment of 129 a.m.u. and trimethyl- silanol from the molecular ion. This ion remains at m/e 215 in the spectrum of the di-d,-TMS ether of XVII, but is shifted to m/e 216 in the spectrum of the 16-d, analogue: in the case of the 15,15,17,17-d4 analogue, 88% of the deuterium atoms were retained in the ion. These results, summarised in Table 2, indicate that the ion of rnje 215 is formed mainly by loss of 129 a.m.u. from ring A (cf ion e) with elimina- tion of trimethylsilanol from ring D.

The presence of a 17cr-alkyl substituent facilitates fragmentation of ring D of the 17~-trimethylsilyloxy steroids, doubtless by promoting initial cleavage of the C-13 to (2-17 bond. Thus, the spectrum of 17cr-methylandrost-5-ene-3/?, 17p-diol TMS ether (VI, Fig. 6) contains ions of m/e 129 and 143 deriving, respectively, from rings A and D.34 The ion of m/e 143 (78%) is much more intense than that of m/e 129 (15%). This is in contrast to the origins of the ions of m/e 129 in the spectra of V, XV, XVI and XVII, which contain no 17-alkyl substituent and which yield ions of m/e 129 mainly by fragmentation of ring A. The base peak (m/e 268) of the spectrum of VI arises from sequential losses of two molecules of trimethylsilanol from the molecular ion.

A free 17-hydroxy group [as in 17a-methylandrost-5-ene-3@, 17p-diol 3-TMS ether (VII, Fig. 7)] directs fragmentation of ring D to a much lesser extent than a trimethylsilyloxy group. The ion of m/e 129, which can arise only from ring A, is again the base peak, and water is eliminated from the molecular ion and certain fragment ions, as observed in the spectrum of IV.

The mass spectra of derived TMS ethers of the trihydroxy analogues are more complex than those of the triols and fragmentation is even more extensive: the base peak (m/e 239) in the spectrum of androst-5-ene-3~,16~,17a-triol TMS ether

TABLE 2. SALIENT MASS SPECTROMETRIC DATA FOR CHARACTERISATION OF THE ION OF mle 215 IN THE SPECTRUM OF XVII AYD OF CORRESPONDING IONS IN THE SPECTRA OF LABELLED ANALOGUES

nz/e 215 216 217 218 219

- - - (XVII) 100 18 d,-TMS 100 15 - 16-dI (99 %) 4 100 18 2 15,15,17,17-d, (61 %) 16 25 30 57 100

- - -

Mass spectra of trimethylsilyl ethers of some A5-3@-hydroxy CIS steroids 937

L U N O

I I I

v)

f In

0 In

N f m -4

0 0 m :I h U 3 z

t

0 0 N -

El

938 C. J. W. BROOKS, D. J. HARVEY, B. S. MIDDLEDITCH and P. VOUROS

(VIII, Fig. 8) contributes only 6.5% of the total ion current. The spectra of VIII, and of the 16a,17P (XVIII), 16j,17v. (XIX) and 16j ,17j (XX)triolsarevery~imilar.~,~

(XX)

In a study of the intramolecular interactions of TMS groups in the formation of rearrangement ions of m/e 147 (p)* from steroid TMS ethers, it was found that the relative abundance of this ion provided an indication of the spatial relationships of the TMS groups.35 Such ions were of relative abundance 70 to 82% in the 70 eV spectra of VIII, XVITI and XX, but are of much lower abundance in the 20eV spectra.

The relatively intense ion of m/e 191 has been observed in the spectra of TMS derivatives of many polyhydroxy including 11,17-diols, 15 ,17-d ioI~ ,~~ 1 6 , 1 7 - d i o l ~ , ~ ~ ~ ~ ' to 40 17,l8-diolsg1 and 15 ,16 ,17 - t r io I~ .~~~~ It has been demonstrated to be a rearrangement ion ( s ) . ~ ~

Me I + ,Me

Me-Si-O=Si I 'Me

(PI mle 147 Me

TMS~=CH-OTMS (4 m/e 191

The formation of the base peak (nzje 239) from VIll is particularly interesting. Metastable evidence attests to its formation from the relatively intense ion of m/e 329. It could be suggested that the latter ion arises simply by sequential loss of trimethyl- siIanol and a fragment of 103 a.m.u. (TMSOCH,.) from the molecular ion. Ions of type [M - 103]+ are typical of TMS ether derivatives of primary aliphatic alcohols44

* These ions may be distinguished from other ions of m/e 147 since they shift to nz/e 162 in the spectra of the corresponding d,-TMS ethers.

Mass spectra of trimethylsilyl ethers of some A5-3B-hydroxy C, , steroids 939

and other primary carbinols such as 21-hydroxy steroids.45 [These ions may also appear in the spectra of TMS ether derivatives of steroids with vicinal hydroxyl groups.40 In the spectra of VIII, XVIII, XIX and XX, however, the ion corresponding in nominal mass to [M - 103]+ is merely the second isotope peak of the ion of m/e 417 (formed by loss of a methyl radical from [M - 90]+.)]. By selectively labelling the 16- and 17-trimethylsilyloxy groups with l8O, we have shown that neither is present in the ion of m/e 329.32 Spectra of 16-d1, 174, and 15,15,16-d, analogues of these compounds show that this ion is formed by more than one route (Table 3). In fact, evidence from metastable ions indicates that the ion of m/e 329 is formed both from the molecular ion and from that of type [M - 90]+.. The major route to the ion of nzje 329 is by loss from the molecular ions of two trimethylsilyloxy groups (from C-16 and C-17), hydrogen atoms from C-17 and C-15, a hydrogen atom

TABLE 3. SALIENT MASS SPECTRAL DATA FOR CHARACTERISATION OF THE ION OF mle 329 IN TBE SPECTRUM

OF XVIII AND OF CORRESPONDING IONS IN T H E SPECTRA OF LABELLED ANALOGUES

mle 329 330 331 338

(XVII I) d,-TMS

1 7 4 1 (98 X )

16-180

16-dl (997,’)

15,15,16-d3 (86 ”.)

17-’*0

OTMS

OTMS

I OTMS

mle 329

SCHEME 4

940 C. J. W. BROOKS, D. J. HARVEY, B. S . MIDDLEDITCH and P. Vou~os

which may be from C-15, or another carbon atom (not C-16) and a carbon atom (probably C-17). A possible mechanism is shown in Scheme 4. A minor route to the ion of m/e 329 apparently proceeds by loss from the molecular ion of the two trimethylsilyloxy groups, a carbon atom (probably C-16), a hydrogen atom from C-16 and two more hydrogen atoms (not from C-17, and only partially from C-15). Detailed elucidation of the genesis of the ion of m/e 329 is hindered by the multiplicity of fragmentation routes and the need for more extensive isotope-labelling of atoms remote from the oxygenated functional groups.

It should be noted, that with two trimethylsilyloxy groups in ring D, much of the fragmentation of the molecule is directed from that vicinity. As much as 40 % of the initial loss of trimethylsilanol originates from ring D.33

Fragmentations of compounds with hydroxyl and keto groups in ring D The spectrum of 38-1 7,9-dihydroxyandrost-5-en-16-one TMS ether (IX, Fig. 9)4,8

has an ion of m/e 129 as the base peak and affords many of the expected fragmenta- tions. The spectrum of the 15,15,17-d3 analogue revealed that a proportion of the ions of m/e 129 retained all of the hydrogen atoms at C-15 and C-17. Moreover, it was found that in the spectrum of the d,-TMS ether, only 55 % of the ions of m/e 129 were shifted to m/e 138 (as expected for d) and that the rest were shifted only to m/e 135. It seems likely that the minor component of the base peak is formed by fragmentation of ring D as in Scheme 5. These results are summarised in Table 4.

1 I

Si-Me

Me mle 129

SCHEME 5

Mass spectra of trimethylsilyl ethers of some As-3P-hydroxy C,, steroids

I I

94 f

0

0 m E

c y o m - z

m t- i'i i

+ w

m N

I

0 0

I , ,I

cn E

O% 0 07

F

zt 0 m-

I- 1 U

0 52

942 C . J. W. BROOKS, D. J. HARVEY, B. S. MIDDLEDITCH and P. Vou~os

TABLE 4. SALIENT MASS SPECTRAL DATA FOR CHARACTERISATION OF THE ION OF mle 129 IN THE SPECTRUM OF 1x AND OF CORRESPONDING IONS IN THE SPECTRA OF LABELLED ANALOGUES

inle 129 130 131 132 135 138

- - 100 17 6 . 1 - - 2 2 76 100

(IX) &TMS

- 15,15,17-d3 (50 %) 100 35 65 60 3

The weak ion of type [M - 56]+. at mje 392 was found not to be formed by fragmenta- tion of ring D and is probably formed as for I.

The spectrum of 3/3,16a-dihydroxyandrost-5-en-17-one TMS ether (X, Fig. 10)438 has been described in detai1.10,32 l80-Labelling at C-17, and deuterium labelling at C-16 have shown that the abundant ion of m/e 304 is formed by fragmentation of ring D,8,1° as shown in Scheme 6. The base peak at m/e 214 is formed by elimination

I mle 304

-mison I ./-Me

(i) ni/e 214

( j ) mle 199

SCHEME 6

of trimethylsilanol from this ion. A metastable ion attests to the fragmentation of this ion (i) to give the ion of m/e 199 (j). High resolution mass measurements show that the ion of m/e 196 has composition C,,H,,OSi. The spectrum of the d,-TMS ether contains a corresponding ion at mle 205, indicating that it contains one intact TMS group, whereas the ion remains at mje 196 in the spectra of its 16-d1 analogue and 17-180 analogue. The structure and mode of formation of this ion remain to be eluci- dated. The isotope-labelling data are consistent with previous suggestions that the ion of mje 175 is formed by elimination of C-15 to C-17 from e , 4 I 1 O and indicate that the ion of m/e 117 comprises C-15 and C-16, with substituents and an additional hydrogen atom. Partial high-resolution mass spectra of IX and X, showing composi- tions of salient ions, are presented in Table 5.

Mass spectra of trimethylsilyl ethers of some A6-3/3-hydroxy C1, steroids 943

R N

Y

0 0 N

m N

+8

0

0 0 In

0 0 *

CD U r 4

m LD

0 0 IY

h c1 e m E h ." P

(cl 0 h

5 0

E e * 0

&

944 C. J. W. BROOKS, D. J. HARVEY, B. S. MIDDLEDITCH and P. Vou~os

TABLE 5. PARTIAL HIGH RESOLUTION SPECTRA OF IX AND X

mle (calc.)

11 7.0736 129.0372 129.073 6 171.1 174 1 96.1 283 199,1487 214.1721 304.2222 433.2594 448.2829

elemental comp.

error (m.m.u.) (1x1 (X)

-1.58 +0.18

+ 1.66 absent -0.01 +0.21 absent +3.37 +2.70

-0.54

-1.65 -1.75 - 1 '70 -0.05 -0.07 -0.45 +0.70 0.00

+ 1.47 +0.71

The spectrum of 3~,11~,16a-trihydroxyandrost-5-en-17-0ne di(3,16) TMS ether (XI, Fig. 11) is similar in many respects to that of X, but with additionat ions due to eliminations of water (cf. IV and VII). It should be noted that XI also gives rise to an ion of m/e 196 : if this corresponds to the ion from X, it evidently does not contain c-11.

Other androstenol TMS ethers The spectrum of 3/3,18-dihydroxyandrost-5-en-l6-one TMS ether (XII, Fig. 12)

contains a relatively intense [M - 30]+. peak, apparently formed by migration of a TMS group and elimination of the elements of This mechanism is formally that of a 'silyl McLafferty' rearrangement, a fragmentation mode which has subsequently been observed for other corn pound^.^' The ion of m/e 248 in the spectrum of XI1 may comprise rings A and B, and may undergo elimination of trimethylsilanol to yield the base peak at m/e 158. Cleavage of C-13/C-18 apparently gives rise to the ion of m/e 103.

The spectrum of 3/3,17/3-dihydroxyandrost-5-en-l I-one TMS ether (XIII, Fig. 13) contains relatively few ions. This appears to be typical of the compounds which contain an 11-keto group (see above). The [M - 56]+. ion at m/e 392 presumably arises by fragmentation of ring A. Again, this fragmentation mode is promoted by the presence of a keto group.

Androst-5-ene-3B,4a, 17B-triol TMS ether (XXI) is unusual in that its spectrum

?TMS

(XXQ

contains ions of m/e 191, m/e 419, [M - 103]+ and m/e 329, [M - 103,90]+, in only low abundance. Such ions are usually found in higher abundance if adjacent trimethyl- silyloxy groups are present in ring D.

Mass spectra of trimethylsilyl ethers of some A6-3/?-hydroxy CI9 steroids 945

946 C. J. W. BROOKS, D. J. HARVEY, B. S. MIDDLEDITCH and P. VOUROS

CONCLUSIONS

The data and interpretations outlined above provide a firmer foundation for the use of mass spectrometry in the identification of A5-androstenols than had hitherto been a ~ a i l a b l e . ~ ~ It is particularly important to realise that the generality of formation of certain ions is limited. The [M - 56]+. ion observed in the spectra of saturated 16- and 17-ketones is formed by fragmentation of ring D. It has now been shown, that in the spectra of the corresponding A5-androstenols, there is an ion of type [M - 56]+. that originates by fragmentation of ring A. This could be most mis- leading if a compound such as XI11 were to be characterised. Many steroids (and other compounds49) with two or more trimethylsilyloxy groups in close proximity yield ions of m/e 191. Such an ion is not, however, significantly present in the spectrum of XXI. Ions of type [M - 103]+ have been observed in spectra of steroids containing vicinal trimethylsilyloxy groups, but these too are not universally apparent. An ion of m/e 117 is usually indicative of a 20-trimethylsilyloxy moiety of a steroid. This ion is also observed in the spectrum of the 16-trimethylsilyloxy 17-keto steroid (X). While the ion of m/e 129 is indicative of the A5-3/3-trimethylsilyloxy structure, ions of this mass are also formed by fragmentation of ring D of 16-keto-l7-trimethylsilyloxy compounds (Scheme 5). In spectra of d9-TMS ethers, the former shift to m/e 138 and the latter only to m/e 135.

The majority of the spectra of the androstenol TMS ethers discussed above are sufficiently well characterised to permit their analytical use, but some groups of isomers give very similar spectra. In certain cases, gas chromatography is useful in distinguishing isomers, as seen in Table 6. Although 16- and 17-ketones are not easily distinguished using g.c.-m.s. , they may be readily identified by in transitu deuterium labelling: the 16-ketone can take up four deuterium atoms and the 17- ketone only

EXPERIMENTAL Mass spectra (low and high resolution) were obtained as previously reported.51 TMS ethers were prepared by standard techniques and d,-TMS ethers by using d,,-BSA.52 The

method for in tvunsitu deuterium labelling has been described.50 Other deuterium labelling was

TABLE 6 . RETENTION INDICES OF A5-ANDROSTEN-3p-OL TMS ETHERS 6 ft I % OV-1 ,200"

Substituents in parent steroid (1)

16-keto 17-keto 11,17-diketo 16u-hydroxy 17whydroxy l7p-hydroxy 16m, 17u-dihydroxy 16/3,17P-dihydroxy 17p-hydrox y- 17 u-methy 1 17p-hydroxy-17cr-methyl 16u-hydroxy-17-keto 17B-hydroxy-16-keto

2560 2555 2630 2595 2590 2640 2830 2885 2730 2610 2760 2780

a mOnO(3) TMS

Mass spectra of trimethylsilyl ethers of some A5-3p-hydroxy CIS steroids 947

performed by previously reported of ketones in '*O-enriched water.63

lsO-labelled compounds were prepared by exchange

Acknowledgments-This work was partially supported by grants from the M.R.C., S.R.C. (B/SR/ 2398), N. I. H. (GM-13901, GM-16216) and Robert A. Welch Foundation (4-125). We are indebted to J. Watson and I. Sangster for their earlier work on the project, to R. W. Kelly, P. J. Sykes and F. L. Mitchell for provision of compounds XII and XXI, to Miss P. Crain for obtaining high reso- lution mass spectra, and to Mrs S. Sloan for preparation of some of the labelled compounds. We thank M. G. Horning for her interest in and encouragement of this work.

R E F E R E N C E S

1. E. Heftmann, Steroid Biochemistry, Academic Press, New York, 1970. 2. L. F. Fieser and M. Fieser, Steroids, Reinhold, New York, 1959, pp. 524 to 525. 3. C. J. W. Brooks, E. M. Chambaz, W. L. Gardiner and E. C. Horning, Excerpta Med. Int.

4. C. H. L. Shackleton, R. W. Kelly, P. M. Adhikary, C. J. W. Brooks, R. A. Harkness, P. J. Sykes

5. E. M. Chambaz, C. J. W. Brooks, M. G. Horning, E. C. Horning and R. M. Hill, Compt. Rend.

6. M. G. Horning, E. M. Chambaz, C. J. W. Brooks, A. M. Moss, E. A. Boucher, E. C. Horning

7. C. H. L. Shackleton, J.-A. Gustafsson and J. Sjovall, Steroids 15, 131 (1970). 8. J.-A. Gustafsson, C. H. L. Shackleton and J. Sjovall, Eur. J. Biochem. 10, 302 (1969). 9. J.-A. Gustafsson, C. H. L. Shackleton and J. Sjovall, Acta Endocrinol. 65, 18 (1970).

Congr. Ser. 132, 366 (1966).

and F. L. Mitchell, Steroids 12, 705 (1968).

268D, 2817 (1969).

and R. M. Hill, Anal. Biochem. 31, 512 (1969).

10. A. L. Siege], H. Adlercreutz and T . Luukkainen, Ann. Med, Exp. Biol. Fenniai (Helsinki) 47,

11. T. Laatikainen, Steroids 15, 139 (1970). 12. R. Vihko, Acta Endocrinol. Suppl. 109, 1 (1966). 13. J. Sjovall and R. Vihko, Steroids 6, 597 (1965). 14. J. Sjovall and R. Vihko, Steroids 7, 447 (1966). 15. J. Sjovall and R. Vihko, Acta Endocrinol. 57,247 (1968). 16. 0. Janne, J. Perheentupa and R. Vihko, J . Clin. Endocrinol. Merab. 31, 162 (1970). 17. M. E. Manson, C. H. L. Shackleton, F. L. Mitchell, J.-A. Gustafsson and J. Sjovall, Steroids 18,

18. C. H. L. Shackleton, F. L. Mitchell, J.-A. Gustafsson and J. Sjovall, FEBS Letters 11,129 (1970). 19. I. Bjorkhem, J.-A. Gustafsson and S. A. Gustafsson, Eur. J. Biochem. 16, 557 (1970). 20. P. Eneroth, H. Ferngren, J.-& Gustafsson, B. Ivemark and A. Stenberg, Acta Endocrinol.

70, 113 (1972). 21. F. L. Mitchell and C. H. L. Shackleton in 0. Bodansky and C. P. Stewart (Eds.), Advances in

Clinical Chemistry, Vol. 12, Academic Press, New York, 1969, p. 141. 22. C. J. W. Brooks, E. C. Horning and J. S . Young, Lipids 3,391 (1968). 23. J. Diekman and C. Djerassi, J . Org. Chem. 32, 1005 (1967). 24. C. J. W. Brooks, Process Biochem. 2 , 27 (1967). 25. M. Spiteller-Friedmann and G. Spiteller, Fortschr. Chem. Forsch. 12, 440 (1969). 26. L. TokBs, G. Jones and C. Djerassi, J. Amer. Chem. Soc. 90, 5465 (1968). 27. C. C. Sweeley, W. H. Elliott, I. Fries and R. Ryhage, Anal. Chem. 38, 1549 (1966). 28. R. Ryhage and S. Wikstrom, Sci. Tools 14, 1 (1967). 29. R. Blomstrand and J. Gurtler, Arkiv Kemi 30, 213 (1969). 30. L. TokBs, R. T . LaLonde and C. Djerassi, J. Org. Chem. 32, 1012 (1967). 31. C. J. W. Brooks, D. J. Harvey and B. S . Middleditch, J. Org. Chem. 37, 3365 (1972). 32. P. Vouros and D. J. Harvey, Org. Mass Spectrom. 6, 953 (1972). 33 . P. Vouros and D. J. Harvey, Chem. Commun. 765 (1972). 34. B. S. Middleditch, P. Vouros and C. J. W. Brooks, J. Pharm. Pharmacol. 25, 143 (1973). 35. S. Sloan, D. J. Harvey and P. Vouros, Org. Mass Spectrom. 5, 789 (1971). 36. C. J. W. Brooks and B. S. Middleditch in E. Heftmann, (Ed.), Modern Methods of Steroid

22 (1969).

51 (1971).

Analysis, Academic Press, New York, in press.

948 C. J. W. BROOKS, D. J. HARVEY, B. S. MIDDLEDITCH and P. Vou~os

37. J.-A. Gustafsson and J. Sjovall, Eur. J . Biochem. 6, 227 (1968). 38. J.-A. Gustafsson, B. P. Lisboa and J. Sjovall, Eur. J . Biochem. 5, 437 (1968). 39. G. Zucconi, B. P. Lisboa, E. Simonitsch, L. Roth, A. A. Hagen and E. Diczfalusy, Acta Endo-

40. B. P. Lisboa, J.-A. Gustafsson and J. Sjovall, Eur. J . Biochem. 4, 496 (1968). 41. J.-A. Gustafsson and B. P. Lisboa, Steroids 15, 723 (1970). 42. R. W. Kelly, J . Chromatog. 54, 345 (1971). 43. J.-A. Gustafsson, R. Ryhage, J. Sjovall and R. M. Moriarty, J . Amer. Chem. SOC. 91, 1234

44. A. G. Sharkey, R. A. Friedel and S . H. Langer, Anal. Chem. 29, 770 (1957). 45. J.-A. Gustafsson and J. Sjovall, Eur. J . Biochem. 6, 236 (1968). 46. T. Laatikainen and R. Vihko, Steroids 13, 615 (1969). 47. W. P. Weber, R. A. Felix and A. K. Willard, J . Amer. Chem. SOC. 92,1420 (1970). 48. G. von Unruh and G. Spiteller, Tetrahedron 26, 3303 (1970). 49. B. S. Middleditch and D. M. Desiderio, submitted. 50. G. M. Anthony and C. J. W. Brooks in R. Stock (Ed.), Gas Chromatography 1970, Institute of

51. C. J. W. Brooks, B. S. Middleditch and D. J. Harvey, Org. Muss Spectvom. 5 , 1429 (1971). 52. J. A. McCloskey, R. N. Stillwell and A. M. Lawson, Anal. Chem. 40,233 (1968). 53. A. M. Lawson, F. A. J. M. Leemans and J. A. McCloskey, Steroids 14,603 (1969).

crinol. 56, 413 (1967).

(1968).

Petroleum, London, 1971, p. 70.