LC-MS/MS method for the quantification of new psychoactive substances and evaluation of their urinary detection in humans for doping control analysis.

The constant legal adaptation of new psychoactive substances (NPS), challenges their evaluation in different fields. In sports, NPS are prohibited in competition with a reporting limit (RL) of 50 ng/mL for the parent compound or a metabolite. However, there is a lack of comprehensive methodologies and excretion studies for monitoring NPS. This work aims to develop an analytical methodology for the NPS quantification and to evaluate the suitability of monitoring the urinary parent stimulants after NPS misuse. A method for the quantification of 14 common NPS was developed and validated. The method was found to be linear in the range 1-1000 ng/mL, and was shown to be accurate and precise. A lowest limit of quantification (LLOQ) of 1 ng/mL was established for all analytes except for benzylpiperazine (5 ng/mL). The method was able to confirm the identity of the analytes at the LLOQ for most NPS. The methodology was applied to the quantification of the parent compound in urine samples collected from an observational study where several healthy volunteers (n ≥ 6 per drug) ingested active doses of mephedrone (MEPH), methylone (MDMC), 2,5-dimetoxy-4-ethylphenetylamine (2C-E), or 6-(2-aminopropyl)benzofuran (6-APB). It was observed that for MDMC and 6-APB, the quantification of the urinary parent drug at the current RL is a proper strategy for detecting their misuse. However, this strategy seems to be insufficient for evaluating MEPH and 2C-E misuse. Monitoring the most abundant metabolite of MEPH (4'-carboxy-MEPH) and the reduction of the RL to 10 ng/mL for the 2C-E evaluation are proposed.