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

Abstract

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.

Detection time comparison of non‐hydrolysed sulphated metabolites of metenolone, mesterolone and 17α‐methyltestosterone analysed by four different mass spectrometric techniques

“…The second one was a mono‐sulphated metabolite that had the same mass as S‐Mest‐5 and S‐Mest‐6, but it could not be determined which one it was. A recent publication 23 showed through fraction collection and GC‐EI‐QTOF‐MS analysis that S‐Mest‐5 is the one that was previously identified by Polet et al 21 …”

“…Four non‐hydrolysed sulphated metabolites were identified by Fragkaki et al 28 (listed in Table 1) using LC‐MS and out of those four, S‐Met‐PC, S‐Met‐1 and 3ζ‐sulphate‐1β‐methyl‐5α‐androstan‐17‐one (S‐Met‐3) were also identified using GC‐CI‐QQQ‐MS 26 . A recent publication 23 additionally identified the last one, S‐Met‐2, on GC‐EI‐QTOF‐MS where due to the alternative sulphate cleaving it results in an atypical molecular ion, [M‐SO 4 ] •+ instead of the more common [M‐H 2 SO 4 ] •+ . This explains why it was probably overlooked during the initial investigation 26 but could be identified in the one currently being presented.…”

“…A single LC‐ESI‐QQQ‐MS method was used in all cases, and the targets are listed in Table 1 and depicted in Figure 1. A recent publication 23 identified sulphated metabolites on GC‐MS that had been discovered on LC‐MS but were not reported yet by GC‐MS as either they exhibited alternative sulphate cleaving behaviour or had previously not been analysed in their non‐hydrolysed form on GC‐MS. The metabolites identified in that paper and others were identified on both GC‐EI‐ and GC‐CI‐QQQ‐MS and the transitions added to the pre‐existing methods, listed in Table 2 and depicted in Figure 1, as the intent was to mimic an authentic ITP.…”

“…This indicates that the mass analyser itself can have a great impact on the importance of the detection capability, irrespective of chromatography or ionisation type. Interestingly, S‐Met‐2, the metabolite that was located on GC‐EI‐QTOF‐MS recently because of alternative behaviour, 23 provides the longest detection time of the sulphated and hydrolysed metabolites on GC‐EI‐ and GC‐CI‐QQQ‐MS. This highlights the importance of determining metabolites identified as LTM on LC‐MS and then pair it with the corresponding peak on GC‐MS.…”

“…The reasoning was that these are instances where the non‐hydrolysed sulphated metabolites have demonstrated to provide equal or extended detection time compared to the conventional markers. Recently, work was conducted to identify non‐hydrolysed sulphated metabolites on GC‐MS that had been detected using LC‐MS but not on GC‐MS 23 . This allowed comparing the detection time of the same non‐hydrolysed sulphated metabolites across all four analytical techniques that were selected for this work.…”