Constant Ion Loss Method for the Untargeted Detection of Bis-sulfate Metabolites

Steroid detection and identification remain key issues in toxicology, drug testing, medical diagnostics, food safety control, and doping control. In this study, we evaluate the capabilities and usefulness of analyzing non‐hydrolyzed sulfated steroids with gas chromatography−mass spectrometry (GC–MS) instead of the conventionally applied liquid chromatography−mass spectrometry (LC–MS) approach. Sulfates of 31 steroids were synthesized and their MS and chromatographic behavior studied by chemical ionization−GC−triple quadrupole MS (CI−GC‐TQMS) and low energy−electron ionization−GC−quadrupole time‐of‐flight−MS (LE−EI−GC−QTOF−MS). The collected data shows that the sulfate group is cleaved off in the injection port of the GC–MS, forming two isomers. In CI, the dominant species (ie, [MH – H2SO4]+ or [MH – H4S2O8]+ for bis‐sulfates) is very abundant due to the limited amount of fragmentation, making it an ideal precursor ion for MS/MS. In LE−EI, [M – H2SO4].+ and/or [M – H2SO4 – CH3].+ are the dominant species in most cases. Based on the common GC–MS behavior of non‐hydrolyzed sulfated steroids, two applications were evaluated and compared with the conventionally applied LC–MS approach; (a) discovery of (new) sulfated steroid metabolites of mesterolone and (b) expanding anabolic androgenic steroid abuse detection windows. GC–MS and LC–MS analysis of non‐hydrolyzed sulfated steroids offered comparable sensitivities, superseding these of GC–MS after hydrolysis. For non‐hydrolyzed sulfated steroids, GC–MS offers a higher structural elucidating power and a more straightforward inclusion in screening methods than LC–MS.

Section: Resultsmentioning

confidence: 85%

Section: Resultsmentioning

confidence: 89%

Section: Resultsmentioning

confidence: 99%

Section: Synthesis Of Steroid Sulfatesmentioning

confidence: 99%

Section: Searching For Sulfated Steroid Metabolites Of Mesterolone mentioning

confidence: 99%

See 3 more Smart Citations

Gas chromatography−mass spectrometry analysis of non‐hydrolyzed sulfated steroids by degradation product formation

Polet

Gansbeke

Albertsdóttir

et al. 2019

Liquid chromatography–mass spectrometry behavior of Girard's reagent T derivatives of oxosteroid intact phase II metabolites for doping control purposes

Kiousi

Fragkaki

Kioukia‐Fougia

et al. 2021

Intact phase II steroid metabolites have poor product ion mass spectra under collision‐induced dissociation (CID) conditions. Therefore, we present herein the liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/(MS)) behavior of intact phase II metabolites of oxosteroids after derivatization. Based on the fact that Girard's reagent T (GRT), as derivatization reagent, was both convenient and efficient in terms of the enhancement in the ionization efficiency and the production of diagnostic product ions related to the steroid moiety, the latter was preferably selected between methoxamine and hydroxylamine upon the model compounds of androsterone glucuronide and androsterone sulfate. Sixteen different glucuronides and 29 sulfate conjugated metabolites of anabolic androgenic steroids (AASs), available either as pure reference materials or synthesized/extracted from administration studies, were derivatized with GRT, and their product ion spectra are presented. Product ion spectra include in all cases high number of product ions that in some cases are characteristic for certain structures of the steroid backbone. More specifically, preliminary results have shown major differences in fragmentation pattern for 17α/17β‐isomers of the sulfate conjugates, but limited differentiation for 17α/17β‐isomers of glucuronide conjugates and for 3α/3β‐ and 5α/5β‐stereoisomers of both sulfate and glucuronide conjugates. Further to the suggestion of the current work, application on mesterolone administration studies confirmed—according to the World Anti‐Doping Agency (WADA) TD2015IDCR—the presence of seven intact phase II metabolites, one glucuronide and six sulfates with use of LC–ESI–MS/(MS).

Evaluation of alternative gas chromatographic and mass spectrometric behaviour of trimethylsilyl‐derivatives of non‐hydrolysed sulfated anabolic steroids

Albertsdóttir

Gansbeke

Eenoo

et al. 2023

Sulfated metabolites have shown to have potential as long‐term markers (LTMs) of anabolic–androgenic steroid (AAS) abuse. The compatibility of gas chromatography–mass spectrometry (GC–MS) with trimethylsilyl (TMS)‐derivatives of non‐hydrolysed sulfated steroids has been demonstrated, where, after derivatisation, generally, two closely eluting isomers are formed that both have the same molecular ion [M‐H2SO4]•+. Sulfated reference standards are in limited commercial availability, and therefore, the current knowledge of the GC–MS behaviour of these compounds is mainly based on sulfating and analysing the available standard reference material. This procedure can unfortunately not cover all of the current known LTMs as these are often not available as pure substance. Therefore, in theory, some metabolites could be missed as they exhibit alternative behaviour. To investigate the matter, in‐house sulfated reference materials that bear resemblance to known sulfated LTMs were analysed on GC–MS in their TMS‐derivatised non‐hydrolysed state. The (alternative) gas chromatographic and mass spectrometric behaviour was mapped, evaluated and linked to the corresponding steroid structures. Afterwards, using fraction collection, known sulfated LTMs were isolated from excretion urine to confirm the observed findings. The categories that were selected were mono‐hydroxy‐diones, 17‐methyl‐3,17‐diols and 17‐keto‐3,16‐diols as these are commonly encountered AAS conformations. The ability to predict the GC–MS behaviour of non‐hydrolysed sulfated AAS metabolites is the corner stone of finding new metabolites. This knowledge is also essential, for example, for understanding AAS detection analyses, for the mass spectrometric characterization of metabolites of new designer steroids or when one needs to characterize an unknown steroid structure.