Institute of Pharmacy, Freie Universität Berlin, Königin-Luise-Str. 2+4, Berlin, 14195, Germany.
Laboratorio Antidoping FSMI, Largo Giulio Onesti 1, Rome, 00197, Italy.
Rapid Commun Mass Spectrom. 2020 Dec 30;34(24):e8937. doi: 10.1002/rcm.8937.
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.
Established derivatization protocols to introduce [ H ]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 H O to obtain derivatives doubly labelled with [ H ]TMS and O. 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.
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 (CH ) SiO. Further common fragments were also elucidated.
With the help of stable isotope labelling, the structures of postulated diagnostic fragment ions for the different steroidal subclasses were elucidated. O-labelling of the other compounds will be addressed in future studies to substantiate the obtained findings. To increase method sensitivity MS may be suitable in future bioanalytical applications requiring discrimination of the 2- and 4-hydroxy compounds.
芳香酶抑制剂福美司坦（4-羟基雄甾-4-烯-3,17-二酮）被世界反兴奋剂机构列入运动禁药清单。然而，它也以内源性的形式存在，其 2-、6-和 11-羟基异构体也是如此。本研究的目的是使用气相色谱/电子电离质谱（GC/EI-MS）区分不同的异构体，以提高检测的置信度和选择性。
遵循已建立的衍生化方案，引入[H]TMS，生成具有 3-酮-4-烯结构的九种不同羟基类固醇的全氘三甲基硅烷基化和混合氘化衍生物。福美司坦还被标记上 H 2 O，以获得双重标记有[H]TMS 和 18 O 的衍生物。比较了标记和未标记的 TMS 衍生物的 GC/EI-MS 谱。通过高分辨率 MS（GC/QTOFMS）和串联质谱（MS/MS）实验证实了生成碎片离子的建议。
特定于子类的碎片离子包括 m/z 319 用于 6-羟基化合物和 m/z 219 用于 11-羟基化合物。m/z 415、356、341、313、269 和 267 离子表示 2-和 4-羟基化合物。对于它们的区分，m/z 503→269 的跃迁对福美司坦是选择性的。在 2-、4-和 6-羟基类固醇中，TMS 衍生的甲基自由基的裂解和（CH 3 ）SiO 的中性损失导致 TMSO 自由基的丢失。还阐明了其他常见的片段。
借助稳定同位素标记，阐明了不同甾体子类假定诊断性碎片离子的结构。在未来的研究中，将研究其他化合物的 18 O 标记，以证实所获得的发现。为了提高方法的灵敏度，MS 可能适用于未来需要区分 2-和 4-羟基化合物的生物分析应用。
