Elsevier

Journal of Chromatography B

Volume 855, Issue 2, 15 August 2007, Pages 262-270
Journal of Chromatography B

Validated liquid chromatographic–electrospray ionization mass spectrometric assay for simultaneous determination of 3,4-methylenedioxymethamphetamine and its metabolites 3,4-methylenedioxyamphetamine, 3,4-dihydroxymethamphetamine, and 4-hydroxy-3-methoxymethamphetamine in squirrel monkey plasma

https://doi.org/10.1016/j.jchromb.2007.06.034Get rights and content

Abstract

3,4-Methylenedioxymethamphetamine (MDMA) is a recreational drug with neurotoxic potential. Pharmacokinetic data of MDMA and its metabolites may shed light on the mechanism of MDMA neurotoxicity. An LC–MS assay with electrospray ionization (ESI) is presented for quantifying MDMA and its metabolites 3,4-methylenedioxyamphetamine (MDA), 3,4-dihydroxymethamphetamine (HHMA), and 4-hydroxy-3-methoxymethamphetamine (HMMA) in squirrel monkey plasma. The method involved enzymatic conjugate cleavage and protein precipitation. Separation was achieved within 14 min. The method was validated according to international guidelines with respect to selectivity, linearity, accuracy, precision, recovery, and matrix effect. The present method should prove useful for acquiring pharmacokinetic and toxicokinetic data in squirrel monkeys.

Introduction

The drug 3,4-methylenedioxymethamphetamine (MDMA; “Ecstasy”) is a psychotropic agent chemically and pharmacologically related to amphetamine and mescaline [1]. Since the early 1980s, MDMA has gained great popularity as a recreational drug [2], [3]. Abuse of MDMA is associated with the risk of severe, sometimes fatal intoxication [4], [5], [6], [7]. In addition, there is considerable evidence to indicate that MDMA has neurotoxic potential toward brain serotonergic and/or dopaminergic nerve terminals [1], [2], [3], [8], [9], [10]. In rats, squirrel monkeys, rhesus monkeys and baboons, MDMA affects primarily serotonergic nerve terminals. In mice, MDMA produces selective toxic effects on dopaminergic nerve endings. Which animal model best predicts neurotoxic effects in humans is not presently known. However, determination of metabolite formation and calculation of pharmacokinetic and toxicokinetic data of MDMA and its main metabolites in various species may shed light on mechanisms of MDMA neurotoxicity [11].

Systemic metabolism of MDMA may play a role in MDMA neurotoxicity. This is suggested by the observation that direct injection of MDMA into the brain fails to reproduce the neurotoxic effects seen after systemic MDMA administration [12], and the report that alteration of cytochrome P450-mediated MDMA metabolism influences MDMA-induced neurotoxicity [13]. Species differences in neurotoxicity profile (rat/squirrel monkey: serotonin; mouse: dopamine) also suggest that species differences in drug metabolism may influence the neurotoxic potential of MDMA. Indeed, by characterizing the formation of various MDMA metabolites in different animal species (rat, mouse, and squirrel monkey), it may be possible to gain insight into mechanisms of MDMA neurotoxicity.

MDMA metabolism proceeds via two pathways, which operate in unison but at different rates, depending on species. The first involves demethylenation to 3,4-dihydroxymethamphetamine (HHMA) followed by O-methylation to 4-hydroxy-3-methoxymethamphetamine (HMMA) and O-conjugation with sulfate or glucuronic acid. The second entails initial N-demethylation to 3,4-methylenedioxyamphetamine (MDA), followed by deamination and oxidation to the corresponding benzoic acid derivatives conjugated with glycine [14]. In primates (humans, squirrel monkeys), the first pathway appears to predominate, whereas in rodents (rats, mice), the second pathway is more active (although both pathways are operant in all species). Metabolites of MDMA such as HHMA and 3,4-dihydroxyamphetamine (HHA) are easily oxidized to their corresponding quinones which, in turn, can form adducts with glutathione and other thiol-containing compounds [15], [16], [17]. Recently, such adducts have been implicated in MDMA neurotoxicity [18].

Because of the physicochemical properties of HHMA, extraction from biological samples for simultaneous analysis with MDMA and its less polar metabolites (MDA, HMMA) is difficult. Indeed, to date, only one method for the simultaneous determination of the enantiomers of MDMA and its metabolites HHMA, HMMA, and MDA has been published. Unfortunately, sample preparation for the described GC/MS method is time-consuming and complicated, as it requires a two-step derivatization procedure [19]. We now describe a simple LC–electrospray ionization (ESI)-MS method for simultaneous quantification of MDMA, HHMA, HMMA, and MDA in squirrel monkey plasma.

Section snippets

Chemicals and reagents

Methanolic solutions (1000 mg/l) of racemic HMMA and methanolic solutions (100 mg/l) of racemic MDMA-d5 and MDA-d5 were obtained from Cerilliant (Round Rock, TX, USA). Methanolic solution of racemic MDMA and MDA were obtained from Lipomed (Cambridge, MA, USA). 4-Hydroxymethamphetamine (pholedrine), 4-methylcatechol, ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA), and glucuronidase type HP-2 from helix pomatia (glucuronidase activity ≥100.000 units/ml and sulfatase activity ≤7.500 

Sample preparation

A simple sample preparation involving protein precipitation with PCA was performed to measure the analytes in a small sample (100 μl) of plasma. Because HHMA and HMMA cannot be found in their free form in plasma (and quantification of the conjugates is not possible due to the lack of reference substances), conjugate cleavage was necessary prior to the protein precipitation. The reaction time during enzymatic cleavage could be reduced from 16 h to 90 min by increasing the reaction temperature from

Conclusions

The LC–ESI-MS assay presented here is the first to allow for simultaneous and reliable quantification of the enantiomers of MDMA and its metabolites HHMA, HMMA, and MDA in squirrel monkey plasma. Using this assay, it should be possible to collect pharmacokinetic and toxicokinectic data in MDMA-treated squirrel monkeys. Such data can be compared to that available in humans. By exploring the relationship between plasma levels of MDMA (and its various metabolites) and serotonin neurotoxicity in

Acknowledgements

The authors thank Armin A. Weber for his technical support and George Hatzidimitriou for his support with the animal work. This work was supported by PHS Grants DA05707 and DA017964.

References (30)

  • K.M. Hegadoren et al.

    Neurosci. Biobehav. Rev.

    (1999)
  • C. Lora-Tamayo et al.

    Forensic Sci. Int.

    (1997)
  • H.H. Maurer et al.

    Toxicol. Lett.

    (2000)
  • R. de la Torre et al.

    Trends Pharmacol. Sci.

    (2004)
  • F.T. Peters et al.

    Forensic Sci. Int.

    (2007)
  • B. Bandy et al.

    Arch. Biochem. Biophys.

    (2001)
  • C. Hartmann et al.

    J. Pharm. Biomed. Anal.

    (1998)
  • H. Kalant

    Can. Med. Assoc. J.

    (2001)
  • R.H. Schwartz et al.

    Pediatrics

    (1997)
  • B.K. Logan

    J. Anal. Toxicol.

    (2001)
  • A.L. Jones et al.

    Aliment. Pharmacol. Ther.

    (1999)
  • A. Walubo et al.

    Hum. Exp. Toxicol.

    (1999)
  • T.D. Steele et al.

    Addiction

    (1994)
  • U.D. McCann et al.

    Neuropsychopharmacology

    (1994)
  • L.S. Seiden et al.

    NIDA Res. Monogr.

    (1996)
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