Quantification of methylone and metabolites in rat and human plasma by liquid chromatography-tandem mass spectrometry

Methylone is a commonly abused synthetic cathinone derivative marketed as a “legal” alternative to “ecstasy” or cocaine. Previous studies examined the metabolism of methylone in vitro and in vivo; 4-hydroxy-3-methoxymethcathinone (HMMC) was identified as the primary metabolite, with other reported minor metabolites, 3,4-methylenedioxycathinone (MDC) and 3,4-dihydroxymethcathinone (HHMC). However, limited information is known about methylone and its metabolites’ pharmacokinetics. We developed and fully validated a method for the simultaneous quantification of methylone, HMMC, MDC and HHMC by liquid chromatography-tandem mass spectrometry in 100 µl rat and human plasma. β-Glucuronidase was utilized for plasma hydrolysis, followed by perchloric acid protein precipitation and solid-phase extraction utilizing cation exchange columns. Chromatographic separation was performed with a Synergi Polar column in gradient mode, and analytes were determined by two multiple reaction monitoring (MRM) transitions. Linear ranges of 0.5–1,000 µg/l (methylone, HMMC and MDC) and 10–1,000 µg/l (HHMC) were achieved. Bias and imprecision were generally acceptable, although quantification of HHMC exhibited variability (16.2–37 %). Extraction efficiencies and ion suppression were 89.9–104 % (for HHMC, 15.9–16.2 %) and < 11.4 %, respectively. Methylone and metabolites were stable in plasma for 24 h at room temperature, 72 h at 4 °C, and after three freeze–thaw cycles (except for a 60 % HMMC increase). Human and rat plasma were cross-validated, documenting that rat plasma quality control samples were accurately quantified against a human plasma calibration curve (−23.8 to 12 % bias). As proof of method, rat plasma specimens were analyzed pre-injection and after subcutaneous administration of methylone at 6 mg/kg from 15 to 480 min post-dosing. Methylone, HMMC, MDC and HHMC concentrations ranged from 1.1 to 1,310, 11.2 to 194, 1.9 to 152 and 24.7 to 188 µg/l, respectively.