Analysis of buphedrone and Phase I metabolites in mice urine

Synthetic cathinones, such as buphedrone, mephedrone and 3-fluoromethcathinone, are members of a heterogenous family of new psychoactive substances (NPS) that exhibit unique neuropharmacological effects, analogous to amphetamine, cocaine, methamphetamine, and methylenedioxymethamphetamine. Compounds from this class comprise over 130 substances and were first detected in Europe in 2004. Since then, they have rapidly and extensively appeared in the illicit market, surpassing controlled substance legislation and being responsible for many intoxications and overdose deaths worldwide [1]. This situation has fostered the attention of the clinical, forensic and scientific community in order to better understand cathinone metabolism, and find out potential new markers to estimate drug consumption and confirmation of drug use.

The aim of this study was to identify and quantify the major Phase I metabolites of buphedrone in mice urine by HPLC-MS/MS, using an optimized and validated method. Buphedrone and metabolites derived from its N-dealkylation, β-keto reduction and/or 4-aryl hydroxylation were synthesized and characterized in house using spectroscopic techniques such as NMR. Preliminary screening of mice urine was performed in MS full scan mode aiming at searching for precursor ions corresponding to parent drugs and expected metabolites. Identification and quantification of buphedrone and corresponding Phase I metabolites were performed in MRM mode to achieve high selectivity and sensitivity. The most excreted metabolite, during 24 h following exposure to the drug, is that resulting from N-dealkylated reaction (80.3±11.4 µg mL-1), followed by the N-dealkylated alcohol (59.6±4.7 µg mL-1), and by β-keto reduced N-alkylated metabolite (5.65±0.48 µg mL-1). The metabolite resulting from 4-aryl hydroxylation was not identified. It is also observed that buphedrone was eliminated in mice urine in their unchanged form (146.2±14.9 µg mL-1). To the best of our knowledge, this is the most complete study on the metabolism of buphedrone regarding the quantification of metabolites. Future studies should focus on the study of Phase II metabolites in order to find out if some of these compounds are excreted as glucuronide or sulfate conjugates.