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

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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 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.

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Acknowledgments

This research was supported by the Intramural Research Program of the National Institute on Drug Abuse (NIDA), and the National Institute on Alcohol Abuse and Alcoholism (NIAAA), National Institutes of Health (NIH).

Conflict of interest

There are no financial or other relations that could lead to a conflict of interest.

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Authors and Affiliations

  1. Chemistry and Drug Metabolism, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, 251 Bayview Boulevard, Suite 200 Room 05A-721, Baltimore, MD, 21224, USA

    Kayla N. Ellefsen, Marta Concheiro & Marilyn A. Huestis

  2. Program in Toxicology, University of Maryland Baltimore, Baltimore, MD, USA

    Kayla N. Ellefsen

  3. Drug Design and Synthesis Section, Intramural Research Program, National Institute on Drug Abuse and National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Baltimore, MD, USA

    Masaki Suzuki & Kenner C. Rice

  4. Designer Drug Research Unit, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD, USA

    Joshua S. Elmore & Michael H. Baumann

Authors
  1. Kayla N. Ellefsen
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  2. Marta Concheiro
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  3. Masaki Suzuki
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  4. Kenner C. Rice
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  5. Joshua S. Elmore
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  6. Michael H. Baumann
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  7. Marilyn A. Huestis
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Correspondence to Marilyn A. Huestis.

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Ellefsen, K.N., Concheiro, M., Suzuki, M. et al. Quantification of methylone and metabolites in rat and human plasma by liquid chromatography-tandem mass spectrometry. Forensic Toxicol 33, 202–212 (2015). https://doi.org/10.1007/s11419-015-0263-z

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  • DOI: https://doi.org/10.1007/s11419-015-0263-z

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