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

Abstract

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.

Alternative markers for Methylnortestosterone misuse in human urine

“…Parent compound was detectable for a short time window (44 h) in the glucuronide fraction. Interestingly, we obtained the presence of three peaks (RTs) with mass spectra compatible with dehydroxylated metabolite referred initially by Chundela et al 10 Prompt from the recent work of Polet et al 36 we assumed that these peaks may attribute to the direct detection of intact main sulfate metabolite S2. To prove this, the sulfate S2 metabolite was synthesized and analyzed in GC–MS/MS after per TMS derivatization (Figure 4), resulting in three peaks with identical retention times and mass spectra with those obtained from excretion urine samples.…”

“…The potential methylnortestosterone metabolites were analyzed using LC/Q/TOF‐MS in targeted MS/MS mode, monitoring the fragments with m/z 96.9619 (corresponding to the anion [HSO 4 ] − ) and 79.9550 (corresponding to the anion [SO 3 ] − ) from the product ion spectrum of each [M‐H] − . Both fragments were present in product ion spectrum for the three potential sulfate metabolites indicating the presence of sulfate conjugate 36 . No further structural information was revealed through the study of the product ion mass spectra of the tested metabolites because of the lack of any further diagnostic ions.…”

“…LC–MS(/MS) direct detection of intact phase II metabolites 29,30 has widely been applied revealing in metabolites that last in urine for longer periods of time with numerous applications including stanozolol, 18,19 boldione, 31 boldenone, 32 methandienone, 33 methenolone 34 and mesterolone 35 . More recently the direct detection of intact sulfate‐conjugated steroids by GC–MS(/MS) has been reported 36 offering an alternative powerful tool for the detection of sulfo‐conjugated metabolites in doping control applying a rather simple sample preparation procedure.…”

Annual banned‐substance review: Analytical approaches in human sports drug testing 2019/2020

“…Polet et al assessed the potential of targeting intact sulfoconjugates of AAS metabolites of mesterolone by GC‐MS/MS in routine doping controls. Following LLE, evaporation, and trimethylsilylation, and extracts of pre‐ and post‐administration urine samples were subjected to GC‐chemical ionization (CI)‐MS/MS using in silico‐predicted precursor/product ion pairs and GC‐ low energy EI‐Q/TOF for structural characterization 63 . Chromatography was conducted using a fused silica capillary column of 12 m × 0.25 mm (inner diameter) dimension, exhibiting a film thickness of 0.25 μm or, alternatively, 15 m × 0.2 mm (inner diameter) with 0.11 μm film thickness.…”

The 37th Manfred Donike workshop on doping analysis

“…Combined with conventional or high resolution/high accuracy GC–MS approaches, the metabolic fates of 7‐oxo dehydroepiandrosterone and methylstenbolone were studied, yielding relevant information on biotransformation reactions in general and characteristic long‐term metabolites, respectively. Further, the unconventional strategy of measuring (bis)sulfate metabolites by GC–MS(/MS) approaches without hydrolysis of the steroid conjugates was shown to be feasible, and employing low energy electron ionization enabled competitive retrospectivity for sports drug testing purposes as exemplarily shown for phase‐II metabolites generated from mesterolone …”