Short communicationLiquid 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 rat brain
Introduction
3,4-Methylenedioxymethamphetamine (MDMA; “Ecstasy”) is a psychotropic drug which has gained great popularity over the last two decades [1], [2]. MDMA produces a state of consciousness with emotional and sensual overtones [3]. In addition to documented abuse liability [4], MDMA has neurotoxic potential toward brain serotonergic and/or dopaminergic nerve terminals [1], [2], [5], [6], [7], [8]. In rats, as well as in squirrel monkeys, rhesus monkeys and baboons, MDMA produces selective neurotoxic effects on serotonergic nerve terminals. In contrast, in mice, MDMA produces selective toxic effects on dopaminergic nerve endings [9].
Despite considerable research, mechanisms underlying MDMA actions are not fully understood. Some observations, such as dose-dependency, high correlation between MDMA levels and subsequent serotonin neurotoxicity [10] point to (but do not establish) the importance of the parent compound (MDMA), while others suggest a possible role for MDMA metabolites [11]. MDMA metabolism proceeds via two pathways at different rates, depending upon the 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 [12].
To assess the role of the parent compound (MDMA) and/or its metabolites in the biobehavioral effects of MDMA, a method is needed to allow for concomitant measurement of MDMA and its metabolites in specific brain target sites. The rat should be an appropriate animal model for measuring concentrations of MDMA and its metabolites in brain tissue for several reasons: (1) MDMA-induced serotonin neurotoxicity is well documented in the rat [13], (2) the neurotoxic profile of MDMA in rats parallels that in primates [6], (3) the behavioral pharmacology of MDMA in the rat is reasonably well characterized [14], and (4) unlike non-human primates, rats are readily available and ideally suited for studies that require sampling of brain tissue. In the following, a simple LC–electrospray ionization (ESI)-MS method is described for simultaneous quantification of MDMA, HHMA, HMMA, and MDA in brain tissue of rats.
Section snippets
Chemicals and reagents
Chemicals and reagents were the same as described in our previous study [15] and were of analytical grade or highest purity available.
Rat brain tissue samples
Blank rat brain tissue samples were used for validation of the procedure and taken from male Sprague Dawley rats (Harlan, Indianapolis, IN, USA). Rat brain tissue samples for freeze/thaw and proof of applicability experiments were obtained from two different rats previously treated with MDMA or HHMA. The first rat was treated with an 80 mg/kg oral dose of racemic
Sample preparation
Analysis was performed in brain areas rich in 5-HT terminals (cerebral cortex) in which 5-HT deficits are known to be more severe than in regions containing fibers of passage (hypothalamus) or cell bodies (brain stem) [6]. A simple sample preparation involving homogenization of small pieces of brain tissue was performed to measure analytes of interest. Addition of preservatives was necessary to prevent oxidation of HHMA during sample preparation and analysis [20]. Because initial experiments
Conclusions and perspectives
The LC–ESI-MS assay presented here is the first to allow for simultaneous and reliable quantification of MDMA and its metabolites HHMA, HMMA, and MDA in rat brain tissue. This assay should make it possible to explore the relationship between brain concentrations of MDMA and its metabolites and pharmacological and toxic effects of MDMA of interest.
Acknowledgements
The authors thank Armin A. Weber for his technical support. This work was supported by USPHS Grants DA05707 and DA017964.
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Inhibition potential of 3,4-methylenedioxymethamphetamine (MDMA) and its metabolites on the in vitro monoamine oxidase (MAO)-catalyzed deamination of the neurotransmitters serotonin and dopamine
2016, Toxicology LettersCitation Excerpt :It seems unlikely for the rather polar metabolites DHMA, HMMA, and HMA and especially their sulfate and glucuronide conjugates to pass the blood brain barrier. Earlier studies detected only negligible concentrations of DHMA and HMMA in rat brain after conjugate cleavage (Mueller et al., 2008) which should be due to in brain metabolic formation. However, such data in humans are not available limiting knowledge on presence and concentrations of free and/or conjugated MDMA metabolites in the brain.
Neurotoxicity of "ecstasy" and its metabolites in human dopaminergic differentiated SH-SY5Y cells
2013, Toxicology LettersCitation Excerpt :MDMA and its metabolites used in the present study have been quantified in the urine and plasma of human “ecstasy” users (Kolbrich et al., 2008; Pizarro et al., 2004; Segura et al., 2001), but unfortunately their presence in the human brain was yet not reported. Several works found N-Me-α-MeDA in the brain of rats administrated with MDMA via subcutaneous or oral route (Chu et al., 1996; Lim and Foltz, 1988; Mueller et al., 2008). Noteworthy, rats exposed to a binge dose of MDMA tend to accumulate N-Me-α-MeDA conjugates in the brain (Erives et al., 2008).
Sulfation of the 3,4-methylenedioxymethamphetamine (MDMA) metabolites 3,4-dihydroxymethamphetamine (DHMA) and 4-hydroxy-3-methoxymethamphetamine (HMMA) and their capability to inhibit human sulfotransferases
2011, Toxicology LettersCitation Excerpt :Looking at these estimations, a relevant in vivo inhibition of DA sulfation might be expected. It is known that metabolites of MDMA can also be formed in the brain (Mueller et al., 2008b). Recently, it was discussed that a part of the described neurotoxicity of MDMA (Kalant, 2001; Monks et al., 2004; de la Torre et al., 2004; Easton and Marsden, 2006) could be explained by inhibition of the COMT by the catecholic metabolite DHMA in the central nervous system (CNS) (Meyer and Maurer, 2009).
Simultaneous liquid chromatographic-electrospray ionization mass spectrometric quantification of 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) and its metabolites 3,4-dihydroxymethamphetamine, 4-hydroxy-3-methoxymethamphetamine and 3,4-methylenedioxyamphetamine in squirrel monkey and human plasma after acidic conjugate cleavage
2009, Forensic Science InternationalCitation Excerpt :By characterizing the formation of various MDMA metabolites in different species (human, rat, mouse, and squirrel monkey), insight may be gained into processes underlying MDMA neurotoxicity. Also helpful for that matter might be measuring concentrations of MDMA and its metabolites in relevant brain sites [13]. MDMA metabolism proceeds via two pathways at different rates, depending upon the species.
Current status of hyphenated mass spectrometry in studies of the metabolism of drugs of abuse, including doping agents
2012, Analytical and Bioanalytical Chemistry