Mass spectra of trimethylsilyl ethers of some Δ⁵‐3β‐hydroxy C ₁₉ steroids

Abstract

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 NCHOLESTEROL is present in practically all living organisms, and is the primary source of mammalian steroidal hormones. It may, for example, be metabolised to progesterone 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 conjugates) 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.2The 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 eightyear 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 germfree 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.20Despite 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 measurements, have elucidated some routes of fragmentation. 925

Mass spectrometric analysis of androstan-17β-ol-3-one and androstadiene-17β-ol-3-one isomers

“…The fragmentation of 5␣-androstan-17␤-ol-3-one (12) and its analogs (13)(14)(15)(16)(17)(18) was considerably different from those described for analogous steroids that included carbon-carbon double bonds. The product ion mass spectrum°of°the°protonated°molecule°s°of°12 (Figure°2a) demonstrated the efficient dissociation of the precursor ion upon application of a collision offset voltage of 30 V. In contrast to the earlier depicted dissociation patterns, the saturation of the steroid nucleus resulted in an abundant fragment ion at m/z 255 by elimination of two water°molecules° [31].°The°spectrum°was°dominated°by less diagnostic product ions separated by methylene units (14 u).…”

“…The fragmentation route to the ion t at m/z 215 is considered diagnostic for steroids bearing an androstan-17␤-ol-3-one structure. An elimination of water (Ϫ18 u) and acetone (Ϫ58 u) is suggested (Scheme 6), and support for this mechanism was ob-tained by the analysis of the methylated analogs (17)(18) as well as deuterium labeling of 12 yielding the Compounds 14 and 15. The introduction of methyl residues at C-17 or C-1 incremented the fragment ion at m/z 215 to 229, indicating that these positions remain in the product ion after elimination of a total of 76 u from the precursor°ion° (Figure°2b°and°c,°Table°2).°Deuteration°of C-16 and C-17 (15) caused a shift of m/z 215 to 218, providing evidence for the presence of these carbons in the respective product ion, while all four deuterium atoms of 14 located at C-2 and C-4 were eliminated from the°steroid°with°the°loss°of°acetone°(Table°2).°The°release of acetone from 2-and 3-keto steroids has been observed after EI in earlier studies by Budzikiewicz, Djerassi,°and°coworkers° [4,°32],°and°substantial°work has been performed to elucidate the corresponding fragmentation°pathway° [33].°The°participation°of°the hydrogen located at C-6 in the rearrangement has been identified by deuterium labeling, and a comparable dissociation route after ESI and CID of androstan-17␤-ol-3-ones has been proposed in the present study accordingly.…”

Identification of a new inborn error in bile acid synthesis: mutation of the oxysterol 7alpha-hydroxylase gene causes severe neonatal liver disease.

“…The retention index and electron ionization (70 eV) mass spectrum of this compound (Fig. 5) were found to be identical to that of the Me-TMS ether of the authentic monohydroxy bile acid, 3 ␤-hydroxy-5-cholenoic acid (33,(35)(36)(37). The molecular ion was m/z 460, and consistent with a monohydroxy-unsaturated C 24 (Table I).…”

Mass spectrometry in sports drug testing: Structure characterization and analytical assays

“…Although of minor intensity, the fragment ion at m/z 129 is a valuable indicator for a derivatized 3-or 17-hydroxyl function of steroids. The origin of steroidal fragments at m/z 129 has been investigated in detail by analyses of stably deuterated or structurally closely related compounds (Diekman & Djerassi, 1967;Horning, Brooks, & Vanden Heuvel, 1968;Brooks et al, 1973;Masse, Laliberte, & Tremblay, 1985;Rendic, Nolteernsting, & Schänzer, 1999), and the proposed mechanisms of ion formations are shown in Scheme 3a.…”

“…a,b-Unsaturated hydroxysteroids. Various steroid-TMSand steroid-enol-TMS derivatives also generate fragments at m/z 142 and 143 by A-ring cleavage, for example, steroids with a 3-hydroxy-androst-4-ene structure (Brooks et al, 1973) or 3-hydroxy-androst-1-ene nucleus (Table 2, 7) . For the latter in particular, deuterium exchange experiments demonstrated the presence of carbons 2, 3, and 4 in the respective fragment.…”