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
Bibliographic record
Abstract
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 [ 2 H 9 ]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 2 18 O to obtain derivatives doubly labelled with [ 2 H 9 ]TMS and 18 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. 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 (CH 3 ) 2 SiO. 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. 18 O‐labelling of the other compounds will be addressed in future studies to substantiate the obtained findings. To increase method sensitivity MS 3 may be suitable in future bioanalytical applications requiring discrimination of the 2‐ and 4‐hydroxy compounds.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.001 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.001 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.001 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".