Rationale: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. Methods: Stable isotope labelling procedures for the introduction of [ 2 H 9 ]-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). Results: 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 -CH 3 -(CH 3 ) 2 SiH -OH] + . For the different steroid subclasses further diagnostic fragment ions were discussed and structure proposals postulated.Conclusions: Stable isotope labelling of oxo groups as well as derivatization with deuterated TMS groups enables the detection of structure-related fragment ion

Rationale:In various fields of chemical analyses, structurally unknown analytes are considered. Proper structure confirmation may be challenged by the low amounts of analytes that are available, e.g. in early stage drug development, in metabolism studies, in toxicology or in environmental analyses. In these cases, mass spectrometric techniques are often used to build up structure proposals for these unknowns. Fragmentation reactions in mass spectrometry are known to follow definite pathways that may help to assign structural elements by fragment ion recognition. This work illustrates an investigation of fragmentation reactions for gas chromatography/electron ionization mass spectrometric characterization of benzophenone derivatives using the analgesic drug ketoprofen and seven of its related compounds as model compounds. Methods: Deuteration and 18 O-labelling experiments along with high-resolutionaccurate mass and tandem mass spectrometry (MS/MS) were used to further elucidate fragmentation pathways and to substantiate rationales for structure assignments. Low-energy ionization was investigated to increase confidence in the identity of the molecular ion. Results:The high-resolution mass analyses yielded unexpected differences that led to reconsideration of the proposals. Site-specific isotopic labelling helped to directly trace back fragment ions to their respective structural elements. The proposed fragmentation pathways were substantiated by MS/MS experiments. Conclusions:The described method may offer a perspective to increase the level of confidence in unknown analyses, where reference material is not (yet) available.

Rationale The aromatase inhibitor formestane (4‐hydroxyandrost‐4‐ene‐3,17‐dione) is included in the World Anti‐Doping Agency's List of Prohibited Substances in Sport. However, it also occurs endogenously as do its 2‐, 6‐ and 11‐hydroxy isomers. The aim of this study is to distinguish the different isomers using gas chromatography/electron ionization mass spectrometry (GC/EI‐MS) for enhanced confidence in detection and selectivity for determination. Methods Established derivatization protocols to introduce [2H9]TMS were followed to generate perdeuterotrimethylsilylated and mixed deuterated derivatives for nine different hydroxy steroids, all with 3‐keto‐4‐ene structure. Formestane was additionally labelled with H218O to obtain derivatives doubly labelled with [2H9]TMS and 18O. GC/EI‐MS spectra of labelled and unlabelled TMS derivatives were compared. Proposals for the generation of fragment ions were substantiated by high‐resolution MS (GC/QTOFMS) and tandem mass spectrometry (MS/MS) experiments. Results Subclass‐specific fragment ions include m/z 319 for the 6‐hydroxy and m/z 219 for the 11‐hydroxy compounds. Ions at m/z 415, 356, 341, 313, 269 and 267 were indicative for the 2‐ and 4‐hydroxy compounds. For their discrimination the transition m/z 503 → 269 was selective for formestane. In 2‐, 4‐ and 6‐hydroxy steroids loss of a TMSO radical takes place as cleavage of a TMS‐derived methyl radical and a neutral loss of (CH3)2SiO. Further common fragments were also elucidated. Conclusions With the help of stable isotope labelling, the structures of postulated diagnostic fragment ions for the different steroidal subclasses were elucidated. 18O‐labelling of the other compounds will be addressed in future studies to substantiate the obtained findings. To increase method sensitivity MS3 may be suitable in future bioanalytical applications requiring discrimination of the 2‐ and 4‐hydroxy compounds.

The excessive sale of dietary supplements (DSs) has become a global multi-billion market as more and more people turn to DSs for a healthy lifestyle or for aesthetic reasons. DSs are also increasingly popular among athletes; 50-85% of recreational and 35-100% of competitive athletes report taking DSs, the latter more regularly. Unless pathological deficiencies are detected, the intake of DSs for recreational athletes is not recommended. While it may be advisable for competitive athletes to supplement their diet with certain macronutrients (proteins and carbohydrates), many micronutrients (vitamins, minerals) as well as allegedly performance enhancing DSs may only show minimal impact under specific conditions and for certain sports. However, most products lack proof of their effectiveness. In some cases, DSs may even have negative effects and reduce performance. Furthermore, competitive athletes should be aware of the fact that DSs may lead to positive doping tests, as they bear the risk of being contaminated with banned substances, or components may be banned substances themselves. Every single case of taking DSs should therefore be critically assessed and discussed with experts prior to use. DSs cannot replace a balanced diet and hard practice.

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