Mass spectral fragmentation analyses of isotopically labelled hydroxy steroids using gas chromatography/electron...

Gas chromatography coupled to electron ionization mass spectrometry (GC/EI-MS) is used for routine screening of anabolic steroids in many laboratories after the conversion of polar groups into trimethylsilyl (TMS) derivatives. The aim of this work is to elucidate the origin and formation of common and subclass-specific fragments in the mass spectra of TMS-derivatized steroids. Especially in the context of metabolite identification or analysis of designer drugs, isotopic labelling is helpful to better understand fragment ion generation, identify unknown compounds and update established screening methods.

Stable isotope labelling procedures for the introduction of [² H₉ ]-TMS or ¹⁸ O were established to generate perdeuterotrimethylsilylated, mixed deuterated and ¹⁸ O-labelled derivatives for 13 different hydroxy steroids. Fragmentation proposals were substantiated by comparison of the abundances of isotopically labelled and unlabelled fragment ions in unit mass resolution GC/MS. Specific fragmentations were also investigated by high-resolution MS (GC/quadrupole time-of-flight MS, GC/QTOFMS).

Methyl radical cleavage occurs primarily from the TMS groups in saturated androstanes and from the steroid nucleus in the case of enol-TMS of oxo or α,β-unsaturated steroid ketones. Loss of trimethylsilanol (TMSOH) is dependent on steric factors, degree of saturation of the steroid backbone and the availability of a hydrogen atom and TMSO group in the 1,3-diaxial position. For the formation of the [M - 105]⁺ fragment ion, methyl radical cleavage predominates from the angular methyl groups in position C-18 or C-19 and is independent of the site of TMSOH loss. The common [M - 15 - 76]⁺ fragment ion was found in low abundance and identified as [M - CH₃ - (CH₃ )₂ SiH - OH]⁺ . For the different steroid subclasses further diagnostic fragment ions were discussed and structure proposals postulated.

Stable isotope labelling of oxo groups as well as derivatization with deuterated TMS groups enables the detection of structure-related fragment ion generation in unit mass resolution GC/EI-MS. This may in turn allow us to propose isomeric assignments that are otherwise almost impossible using MS only.