In‐depth gas chromatography/tandem mass spectrometry fragmentation analysis of formestane and evaluation of mass spectral discrimination of isomeric 3‐keto‐4‐ene hydroxy steroids

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.