Efficient approach for the detection and identification of new androgenic metabolites by applying SRM GC‐CI‐MS/MS: a methandienone case study

Self Cite

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: 99%

Section: Gc-ms Behavior Of Steroid Sulfatesmentioning

confidence: 99%

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

Polet

Gansbeke

Albertsdóttir

et al. 2019

Self Cite

“…The detection protocol is explained in detail in a previous publication . To summarise: based upon the most common metabolic pathways of AAS in humans, a list of theoretically potential metabolites is generated.…”

Section: Methodsmentioning

confidence: 99%

“…This makes it worthwhile to reinvestigate the metabolism of these two AAS with another soft ionization technique that does not suffer from this ionization bottleneck. The aim of the study reported here was to search for new long‐term metabolites of oxymesterone and mesterolone by application of a gas chromatography–chemical ionization–triple quadrupole mass spectrometry (GC‐CI‐MS/MS) protocol for metabolite detection and identification . CI in combination with triple quadruple technology has proven to significantly increase the sensitivity for a wide range of compounds in comparison with electron ionization (EI) .…”

Section: Introductionmentioning

confidence: 99%

Identification and characterization of novel long‐term metabolites of oxymesterone and mesterolone in human urine by application of selected reaction monitoring GC‐CI‐MS/MS

Polet

Gansbeke

Geldof

et al. 2017

Self Cite

The search for metabolites with longer detection times remains an important task in, for example, toxicology and doping control. The impact of these long-term metabolites is highlighted by the high number of positive cases after reanalysis of samples that were stored for several years, e.g. samples of previous Olympic Games. A substantial number of previously alleged negative samples have now been declared positive due to the detection of various long-term steroid metabolites the existence of which was unknown during the Olympic Games of 2008 and 2012.In this work, the metabolism of oxymesterone and mesterolone, two anabolic androgenic steroids (AAS), was investigated by application of a selected reaction monitoring gas chromatography-chemical ionization-triple quadrupole mass spectrometry (GC-CI-MS/MS) protocol for metabolite detection and identification. Correlations between AAS structure and GC-CI-MS/MS fragmentation behaviour enabled the search for previously unknown but expected AAS metabolites by selection of theoretical transitions for expected metabolites. Use of different hydrolysis protocols allowed for evaluation of the detection window of both phase I and phase II metabolites.For oxymesterone, a new metabolite, 18-nor-17β-hydroxymethyl-17α-methyl-4-hydroxy-androst-4,13-diene-3-one, was identified. It was detectable up to 46 days by using GC-CI-MS/MS, whereas with a traditional screening (detection of metabolite 17-epioxymesterone with electron ionization GC-MS/MS) oxymesterone administration was only detectable for 3.5 days.A new metabolite was also found for mesterolone. It was identified as 1α-methyl-5α-androstan-3,6,16-triol-17-one and its sulfate form after hydrolysis with Helix pomatia resulted in a prolonged detection time (up to 15 days) for mesterolone abuse.

“…Different approaches have been suggested and pursued to accomplish the goal of isolating long‐term metabolites of low abundance and/or unexpected composition in a complex matrix such as urine. One option has been the in silico prediction of structures of potential phase I metabolites of steroidal agents and the analysis of elimination studies with particularly sensitive analytical strategies . As an example, 32 potential metabolites of metandienone were considered for establishing a test method based on GC with chemical ionization (CI) and QqQ analyzer, where the expected precursor‐product ion pairs for each analyte were derived and predicted by structure similarity to earlier studied compounds .…”

Section: Anabolic Agentsmentioning

confidence: 99%

Annual banned‐substance review: analytical approaches in human sports drug testing

Thevis

Kuuranne

Geyer

et al. 2017

There has been an immense amount of visibility of doping issues on the international stage over the past 12 months with the complexity of doping controls reiterated on various occasions. Hence, analytical test methods continuously being updated, expanded, and improved to provide specific, sensitive, and comprehensive test results in line with the World Anti-Doping Agency's (WADA) 2016 Prohibited List represent one of several critical cornerstones of doping controls. This enterprise necessitates expediting the (combined) exploitation of newly generated information on novel and/or superior target analytes for sports drug testing assays, drug elimination profiles, alternative test matrices, and recent advances in instrumental developments. This paper is a continuation of the series of annual banned-substance reviews appraising the literature published between October 2015 and September 2016 concerning human sports drug testing in the context of WADA's 2016 Prohibited List. Copyright © 2016 John Wiley & Sons, Ltd.