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

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 [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.

Fluorovinylsulfones and -Sulfonates as Potent Covalent Reversible Inhibitors of the Trypanosomal Cysteine Protease Rhodesain: Structure–Activity Relationship, Inhibition Mechanism, Metabolism, and In Vivo Studies

“…23.8 kDa) that shows a mass shift corresponding to the mass of the respective compound (Figure ). The observed multiplicity of the peaks was attributed to additions of matrix molecules [ m (sinapinic acid) = 224 Da] to rhodesain, as similar phenomena are described in the literature . The four fluorovinyl derivatives ( 1 , 2a , 2j , and 2k) behaved like the irreversible control substance K11777 , while the non-covalent inhibitor ( 42 ) did not show any detectable adduct signals under the evaluated conditions.…”

“…The resulting spectra consisted of a broad peak corresponding to the protein (ca. 23.3 kDa), − and a second peak corresponding to the covalent protein–inhibitor adduct (ca. 23.8 kDa) that shows a mass shift corresponding to the mass of the respective compound (Figure ).…”

Controlled administration of dehydrochloromethyltestosterone in humans: Urinary excretion and long-term detection of metabolites for anti-doping purpose

“…High resolution accurate mass analyses were performed on an Agilent GC-quadrupol time-offlight (QToF) 7890B/7250 (Agilent Technologies, Milano, Italy), equipped with an Agilent HP1 column (17 m, 0.20 mm, 0.11 µm) with helium as carrier gas as reported earlier [35,36]. Injection was performed in split mode with a 1:10 ratio at 280 °C.…”

Annual banned‐substance review: Analytical approaches in human sports drug testing 2020/2021

“…91 In order to minimize the additional effort of GC/C/IRMS analyses concerning alleged formestane administrations as well as to support attributing atypical or adverse findings to specific drugs, investigations into diagnostic metabolites and metabolic profiles were conducted, which require optimized chromatographic conditions and in-depth knowledge concerning the target analytes' mass spectrometric (dissociation) behavior. In that context, Kollmeier et al 92 In contrast to formestane, arimistane is not known to be of natural/endogenous origin. Hence, the identification of arimistane in urine could be considered as proof of drug use, and analyzing arimistane and its main metabolite androst-3,5-dien-7β-ol-17-one by LC-MS/ MS has been shown to provide superior sensitivity compared to GC-MS(/MS) as reported by Martinez-Brito et al 93 Using postadministration samples collected from three study participants after the oral administration of 25 mg of arimistane, the intact drug was detected up to 10 h (later samples were not analyzed).…”

“…In order to minimize the additional effort of GC/C/IRMS analyses concerning alleged formestane administrations as well as to support attributing atypical or adverse findings to specific drugs, investigations into diagnostic metabolites and metabolic profiles were conducted, which require optimized chromatographic conditions and in‐depth knowledge concerning the target analytes' mass spectrometric (dissociation) behavior. In that context, Kollmeier et al 92 studied the fragmentation pattern of 3‐oxo‐4‐ene‐based steroids with hydroxylations at C‐2, −4, −6, or −11 employing 2 H (via trimethylsilylation) and 18 O labeling and GC–MS/MS analysis. The obtained data suggest the C‐19 methyl group as the predominant leaving group forming [M‐15] + upon EI, and the subsequent dissociation pathways are strongly influenced by the location of the analyte's hydroxylation(s), which allowed for assigning characteristic product ions for the differently hydroxylated species with m/z 319 supporting 6‐hydroxylated analytes, m/z 219 11‐hydroxylated analytes, m/z 415, 356, and others indicating 2‐ and 4‐hydroxylations with the additional precursor/product ion pair m/z 503/269 being specific for formestane.…”