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In this study, we investigate the metabolic profile of strychnine in human urine following controlled administration using liquid chromatography–quadrupole-Orbitrap mass spectrometry. A total of 25 metabolites were characterized and identified. These included 21 previously unreported and 4 previously reported metabolites. Four unreported metabolic pathways were discovered, namely, reduction, methylation, glycosylation, and glucuronidation. Among these, hydroxylation was identified as the major metabolic pathway. The detection windows in the urine for all 25 metabolites were compared with that of the parent drug. Metabolite S10 (2,3-dimethoxy-strychnine) was proposed as a novel potential biomarker for strychnine misuse, rather than strychnine itself, due to its longer detection time and higher number of strychnine-positive time points (exceeding 50 ng/mL in human urine) compared to the parent compound after oral administration. The identification of S10 extends the detection window and offers critical insights for doping control applications.
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