RESUMEN
The study of intact phase II metabolites of endogenous anabolic androgenic steroids (EAAS) gives important information about metabolism and has the potential to improve the detection of doping with testosterone. For analysis with liquid chromatography-mass spectrometry (LC-MS), chemical derivatization at the steroid moiety is a technique to improve the positive ionization efficiency of glucuronidated/sulfated EAAS under collision-induced dissociation (CID) conditions. However, regarding the chromatographic performance, there are still challenges to address, for example, poor peak shape, which is mainly caused by nondefined adsorption in the chromatographic system. Here, we show a novel derivatization technique for the analysis of selected phase II metabolites of EAAS, where the acidic moiety of the glucuronide/sulfate is methylated with different methylation reagents to reduce nondefined adsorption. The methylation reagent trimethylsilyl-diazomethane (TMSD) was preferred over the other tested reagents methyl iodide (MeI) and dimethyl sulfate (DMS). Glucuronidated and sulfated testosterone and epitestosterone were methylated, and their chromatographic performance and CID ion mass spectra obtained in positive ionization mode were investigated. The peak width and peak height were significantly improved for all substances. Methylated testosterone sulfate showed the best results with a 3.5 times narrower peak and 14 times increased intensity compared with underivatized testosterone sulfate. Furthermore, CID ion mass spectra obtained in positive ionization mode showed product ions characteristically for the steroidal backbone for all substances. This preliminary study shows the potential of methylation as a supplementary derivatization technique, which can assist in the development of more sensitive methods due to the improvements in method performance.
RESUMEN
Mephedrone (4-methyl-N-methylcathinone) is a psychostimulant that promotes release of monoamines via the high affinity transporters for dopamine (DAT), norepinephrine (NET) and serotonin (SERT). Metabolic breakdown of mephedrone results in bioactive metabolites that act as substrate-type releasers at monoamine transporters and stereospecific metabolism of mephedrone has been reported. This study compared the effects of the enantiomers of the phase-1 metabolites nor-mephedrone, 4-hydroxytolyl-mephedrone (4-OH-mephedrone) and dihydro-mephedrone on (i) DAT, NET and SERT mediated substrate fluxes, (ii) determined their binding affinities towards a battery of monoamine receptors and (iii) examined the relative abundance of the enantiomers in human urine. Each of the enantiomers tested inhibited uptake mediated by DAT, NET and SERT. No marked differences were detected at DAT and NET. However, at SERT, the S-enantiomers of nor-mephedrone and 4-OH-mephedrone were several times more potent than the corresponding R-enantiomers. Moreover, the R-enantiomers were markedly less effective as releasers at SERT. S-nor-mephedrone displayed moderate affinities towards human alpha1A, human 5-HT2A and rat and mouse trace amine-associated receptor 1. These results demonstrate that stereochemistry dictates the pharmacodynamics of the phase-1 metabolites of mephedrone at SERT, but not at DAT and NET, which manifests in marked differences in their relative potencies, i.e. DAT/SERT ratios. Chiral analysis of urine samples demonstrated that nor-mephedrone predominantly exists as the S-enantiomer. Given the asymmetric abundance of the enantiomers in biological samples, these findings may add to our understanding of the subjective effects of administered mephedrone, which indicate pronounced effects on the serotonergic system.
