In vitro and in vivo metabolism studies of dimethazine

Abstract

The use of anabolic steroids is prohibited in sports. Effective control is done by monitoring their metabolites in urine samples collected from athletes. Ethical objections however restrict the use of designer steroids in human administration studies. To overcome these problems alternative in vitro and in vivo models were developed to identify metabolites and to assure a fast response by anti-doping laboratories to evolutions on the steroid market. In this study human liver microsomes and an uPA(+/+) -SCID chimeric mouse model were used to elucidate the metabolism of a steroid product called 'Xtreme DMZ'. This product contains the designer steroid dimethazine (DMZ), which consists of two methasterone molecules linked by an azine group. In the performed stability study, degradation from dimethazine to methasterone was observed. By a combination of LC-High Resolution Mass Spectrometry (HRMS) and GC-MS(/MS) analysis methasterone and six other dimethazine metabolites (M1-M6), which are all methasterone metabolites, could be detected besides the parent compound in both models. The phase II metabolism of dimethazine was also investigated in the mouse urine samples. Only metabolites M1 and M2 were exclusively detected in the glucuro-conjugated fraction; all other compounds were also found in the free fraction. For effective control of DMZ misuse in doping control samples, screening for methasterone and methasterone metabolites should be sufficient. Copyright © 2016 John Wiley& Sons, Ltd.

Replacing PAPS: In vitro phase II sulfation of steroids with the liver S9 fraction employing ATP and sodium sulfate

“…Previous reports of the equine in vitro phase I metabolism of S provided some comparison with the present study . Several human in vivo and in vitro studies of S metabolism have also been reported . Metabolism of S with UDPGA, ATP, Na 2 SO 4 , and equine liver S9 fraction afforded a range of phase II sulfate and glucuronide metabolites which are summarized in Figure .…”

“…The formation of the 3β‐hydroxy metabolites is known to be favoured in horses, and this is followed by sulfation to give major 3β‐RSS and glucuronylation to give minor 3β‐RSG . Alternatively, the tentative assignment above suggests that formation of the 3α‐hydroxy metabolite 3α‐RS is followed by glucuronylation to give major G12 but not sulfation to 3α‐RSS , in a pattern similar to that observed in humans . Although no unconjugated phase I metabolites were identified directly in this LC–MS study, the phase I metabolism of S is known to afford a number of steroid diol, and triol metabolites which ionize poorly under +ESI conditions .…”

“…Major phase II metabolites were observed including 3β‐RSS which was matched to the available reference material, and a reduced superdrol glucuronide metabolite G12 . Although no reference material was available, G12 was tentatively assigned as 2α,17α‐dimethyl‐5α‐androstane‐3α,17β‐diol 3‐glucuronide (or its 17‐glucuronide regioisomer), as this metabolite did not match the reference material for 3β‐RSG but exhibited similar mass spectrometry behaviour including the proton loss species m/z 495 ([M‐H] − ) and the glucuronide derived fragments m/z 113 (C 5 H 5 O 3 − ), m/z 85 (C 4 H 5 O 2 − ) and m/z 75 (C 2 H 3 O 3 − ) .…”

“…Although no reference material was available, G12 was tentatively assigned as 2α,17α‐dimethyl‐5α‐androstane‐3α,17β‐diol 3‐glucuronide (or its 17‐glucuronide regioisomer), as this metabolite did not match the reference material for 3β‐RSG but exhibited similar mass spectrometry behaviour including the proton loss species m/z 495 ([M‐H] − ) and the glucuronide derived fragments m/z 113 (C 5 H 5 O 3 − ), m/z 85 (C 4 H 5 O 2 − ) and m/z 75 (C 2 H 3 O 3 − ) . A metabolite corresponding to 2α,17α‐dimethyl‐5α‐androstane‐3α,17β‐diol 3‐glucuronide (or its 17‐glucuronide regioisomer) has also been indirectly observed in human in vivo and in vitro studies of S metabolism . Despite this, some caution is required as the formation of stereoisomeric 17β‐methyl‐17α‐hydroxy metabolites through formation and hydrolysis of the tertiary sulfate conjugate provides alternative metabolic pathways .…”

New Potential Biomarker for Methasterone Misuse in Human Urine by Liquid Chromatography Quadrupole Time of Flight Mass Spectrometry

“…Human liver microsomes and an uPA +/+ -SCID chimeric mouse model were employed to produce its different metabolites, and the reduction in C3 together with hydroxylation in C2, C12 (minor), C16 and C20 constitute the main metabolism pathways [10,11,12]. Methasterone and its hydroxylated metabolites were also excreted as glucuronidated compounds, while no sulfated metabolites could be detected in phase II metabolism [13].…”