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

Abstract

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.

In vitro metabolic profile of mexedrone, a mephedrone analog, studied by high‐ and low‐resolution mass spectrometry

“…M2 and M3 with the same m/z 194.1175 and elemental composition C 11 H 15 NO 2 , compatible with the N‐ / O‐ dealkylation of mexedrone. These metabolic reactions (i.e., hydroxylation and N/O ‐dealkylation) are very similar to those already described for similar SCs in vitro (e.g., mephedrone) 41,42 . However, while the carboxylation of the hydroxy‐tolyl portion was reported as the principal urinary marker of intake of mephedrone, 43,44 this metabolic reaction did not seem to take place in our experimental conditions. The extracted ion chromatograms and the related MS spectra for the metabolites of mexedrone are reported in Figure 2.…”

Annual banned‐substance review: Analytical approaches in human sports drug testing 2019/2020

“…The target analytes were chromatographed on a C‐18 analytical column (150 × 2.1 mm, 5 μm particle size) using 0.1% formic acid (solvent A) and acetonitrile (containing 0.1% formic acid, solvent B) before positive ESI and MRM analysis on a QqQ MS. With LODs for methiopropamine and its demethylated analog of 45 and 40 ng/ml, respectively, the detection of the drug administration was accomplished in the 24–36 hr urine fraction, indicating the suitability of these target analytes for doping controls concerning methiopropamine, a fact that was recently called into question regarding octodrine, which was found to be largely metabolized in humans to heptaminol, another stimulant prohibited in sports but not uniquely characteristic for the administration of heptaminol only 158 . Also, new additions to the class of stimulants and established NPS were found to metabolize extensively, 159 and test methods might necessitate adjustments to include recently observed substances and respective characteristic urinary metabolites, for example, from isopropylnorsynephrine (Figure 2) and the consideration of complementary in‐competition testing concerning stimulants using DBS 160 was suggested.…”

“…octodrine, which was found to be largely metabolized in humans to heptaminol, another stimulant prohibited in sports but not uniquely characteristic for the administration of heptaminol only 158. Also, new additions to the class of stimulants and established NPS were found to metabolize extensively,159 and test methods might necessitate adjustments to include recently observed substances and respective…”

Determination of 19 Psychoactive Substances in Premortem and Postmortem Whole Blood Samples Using Ultra-High-Performance Liquid Chromatography–Tandem Mass Spectrometry

“…Only a few screening procedures based on high resolution mass spectrometry have been published in the literature [30][31][32][33][34][35][36]. Whole human blood (premortem and/or postmortem) [37] and urine [38,39] are the biological samples of choice in forensic toxicological analyses [40,41] for several reasons. For postmortem cases, the pathologists try to collect whole blood during autopsies and inspections.…”