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

Abstract

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 chromatographytandem mass spectrometry in 100 ll rat and human plasma. b-Glucuronidase was utilized for plasma hydrolysis, followed by perchloric acid protein precipitation and solidphase 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 lg/l (methylone, HMMC and MDC) and 10-1,000 lg/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 lg/l, respectively.

Pharmacokinetic Profiles and Pharmacodynamic Effects for Methylone and Its Metabolites in Rats

“…However, it is important to note that plasma concentrations of HHMC reported here must be considered semiquantitative owing to variability when detecting this analyte, as reported in the LC-MS/MS method validation (see Ellefsen et al, 2015). C max values for HHMC were 78, 167, and 414 μg/l, while AUC values were 12 200, 30 200, and 86 300 min × μg/l for the 3, 6, and 12 mg/kg methylone doses.…”

“…MDC HCl, HHMC HBr, and HMMC HCl were synthesized as previously described (Ellefsen et al, 2015). [ 3 H]1-methyl-4-phenylpyridinium ([ 3 H]MPP+, specific activity = 85 Ci/ mmol) was purchased from American Radiolabeled Chemicals (St Louis, MO, USA), whereas [ 3 H]serotonin ([ 3 H]5-HT, specific activity = 20 Ci/mmol) was purchased from Perkin Elmer (Shelton, CT, USA).…”

“…Plasma specimens were analyzed using LC-MS/MS, as previously described (Ellefsen et al, 2015). Briefly, 10 μl of β-glucuronidase was added to 100 μl of plasma to hydrolyze metabolite conjugates.…”

“…Importantly, the plasma PK profiles for the metabolites of methylone have not been reported, and the potential bioactivity of methylone metabolites has yet to be systematically evaluated. Ellefsen et al (2015) reported a highly sensitive validated assay for the quantification of methylone, MDC, HHMC, and HMMC using liquid chromatography tandem mass spectrometry (LC-MS/MS) (Ellefsen et al, 2015). In the present study, we utilized this new analytical method to test the hypothesis that methylone is metabolized in rats to form bioactive metabolites.…”

Pharmacokinetic, Ambulatory, and Hyperthermic Effects of 3,4-Methylenedioxy-N-Methylcathinone (Methylone) in Rats

“…The rapid decrease of its dominance in the analytical spectrum at 60 min may be related to its fast conjugation, which would explain its lower plasmatic concentrations compared with nor-methylone. Even though we did not perform enzymatic hydrolysis, we might assume that the rapid decrease of its levels might be related to its fast conjugation with glucuronic and/or sulfuric acid that is typical for fenolic metabolites ( 35 ). Compared with MDMA where peak MDMA concentrations are achieved within 1 h after subcutaneous or oral administration both methylone and its metabolite nor-methylone showed a more rapid kinetic profile ( 36 , 37 ) which is in line with the reported shorter duration of effects in humans (and might lead to more frequent re-dosing by users).…”