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
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.
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