MDMA In Rats Research Articles

Aromatic Bromination Abolishes Deficits in Visuospatial Learning Induced by MDMA ("Ecstasy") in Rats While Preserving the Ability to Increase LTP in the Prefrontal Cortex.

It has recently been demonstrated that aromatic bromination at C(2) abolishes all typical psychomotor, and some key prosocial effects of the entactogen MDMA in rats. Nevertheless, the influence of aromatic bromination on MDMA-like effects on higher cognitive functions remains unexplored. In the present work, the effects of MDMA and its brominated analog 2Br-4,5-MDMA (1 mg/kg and 10 mg/kg i.p. each) on visuospatial learning, using a radial, octagonal Olton maze (4 × 4) which may discriminate between short-term and long-term memory, were compared with their influence on in vivo long-term potentiation (LTP) in the prefrontal cortex in rats. The results obtained indicate that MDMA diminishes both short- and long-term visuospatial memory but increases LTP. In contrast, 2Br-4,5-MDMA preserves long-term visuospatial memory and slightly accelerates the occurrence of short-term memory compared to controls, but increases LTP, like MDMA. Taken together, these data are consistent with the notion that the modulatory effects induced by the aromatic bromination of the MDMA template, which abolishes typical entactogenic-like responses, might be extended to those effects affecting higher cognitive functions, such as visuospatial learning. This effect seems not to be associated with the increase of LTP in the prefrontal cortex.

The role of extracellular serotonin and MDMA in the sensitizing effects of MDMA

MDMA is a non-selective monoamine releasing stimulant with potent serotonergic effects - a pharmacological effect not typically associated with drugs of misuse or efficacious reinforcers. Nonetheless, MDMA is misused by humans and self-administered by laboratory animals. We have previously shown that repeated exposure to MDMA sensitized both the locomotor activating and reinforcing effects of MDMA in rats. Because repeated MDMA exposure often results in decreased markers of serotonin neurotransmission, it is possible that this might underlie the sensitizing effects of MDMA. This was examined in the current study. Male Sprague-Dawley rats were stereotaxically implanted with guide cannula in the medial striatum. They were then pre-treated with saline (n = 11) or MDMA (10 mg/kg, i.p.; n = 10), once daily for five days. Two-days later, all rats received ascending doses of MDMA (0.0, 5.0, 10.0, mg/kg, i.p.) administered at 2 hr intervals, during which locomotor activity was measured and microdialysis samples were collected. Microdialysates were analyzed using liquid chromatography-mass spectrometry and the concentrations of serotonin and MDMA were quantified. Acute MDMA administration produced dose-dependent increases in locomotor activity, which was significantly enhanced by MDMA pre-treatment. Acute MDMA also produced dose-dependent increases in medial-striatal serotonin and MDMA, but this was not impacted by MDMA pre-treatment. These results suggest that the sensitizing effects of MDMA are not due to changes in MDMA-produced synaptic overflow of serotonin in the medial striatum or the absorption/elimination of systemically administered MDMA. More likely candidates are alterations in serotonin receptor mechanisms and/or dopamine neurotransmission following repeated exposure.

Lack of evidence for positive reinforcing and prosocial effects of MDMA in pair-housed male and female rats

3,4-Methylenedioxymethamphetamine (MDMA) is classified as an entactogen, producing feelings of emotional openness and relatedness. One unique feature of MDMA is that people tend to selectively take this drug in social and/or intimate situations. Although MDMA is recognized as having abuse liability, preclinical studies report that it has weak reinforcing effects in animals. The objective of this study was to characterize the positive reinforcing and prosocial effects of MDMA in a translational model of the social environment in which two rats have simultaneous and contingent access to MDMA in close physical proximity. To this end, MDMA self-administration was examined on both fixed and progressive ratio schedules of reinforcement in six groups of rats: (1) isolated males, (2) isolated females, (3) male-male dyads, (4) female-female dyads, (5) male-female dyads, and (6) female-male dyads. For pair-housed rats, data from both rats were analyzed. Next, social preferences were examined in a partner preference test. MDMA failed to produce positive reinforcing effects under all conditions examined. Across a 30-fold dose range (0.01–1.0 mg/kg/infusion), MDMA did not maintain higher responding than saline on both schedules of reinforcement and in all groups tested. In partner preference tests, a history of shared exposure to MDMA did not establish a social preference, and acute administration of MDMA failed to establish a preference for another MDMA-treated rat. These data suggest that social contact does not increase the positive reinforcing effects of MDMA in rats, and that neither contingent nor noncontingent MDMA administration establishes a social preference in rats.

An investigation of the reinforcing effects of MDMA in rats trained to self-administer heroin

An investigation of the reinforcing effects of MDMA in rats trained to self-administer heroin

Differential role of dose and environment in initiating and intensifying neurotoxicity caused by MDMA in rats

BackgroundMDMA causes serotonin (5-HT) syndrome immediately after administration and serotonergic injury in a few days or weeks. However, a serotonin syndrome is not always followed by serotonergic injury, indicating different mechanisms responsible for two adverse effects. The goal of present study was to determine causes for two adverse events and further test that dose and environment have a differential role in initiating and intensifying MDMA neurotoxicity.MethodsInitiation and intensification were examined by comparing neurotoxic effects of a high-dose (10 mg/kg × 3 at 2 h intervals) with a low-dose (2 mg/kg × 3) under controlled-environmental conditions. Initiation of a serotonin syndrome was estimated by measuring extracellular 5-HT, body-core temperature, electroencephalogram and MDMA concentrations in the cerebrospinal fluid, while intensification determined in rats examined under modified environment. Initiation and intensification of the serotonergic injury were assessed in rats by measuring tissue 5-HT content, SERT density and functional integrity of serotonergic retrograde transportation.ResultsBoth low- and high-dose could cause increases in extracellular 5-HT to elicit a serotonin syndrome at the same intensity. Modification of environmental conditions, which had no impact on MDMA-elicited increases in 5-HT levels, markedly intensified the syndrome intensity. Although either dose would cause the severe syndrome under modified environments, only the high-dose that resulted in high MDMA concentrations in the brain could cause serotonergic injury.ConclusionOur results reveal that extracellular 5-HT is the cause of a syndrome and activity of postsynaptic receptors critical for the course of syndrome intensification. Although the high-dose has the potential to initiate serotonergic injury due to high MDMA concentrations present in the brain, whether an injury is observed depends upon the drug environment via the levels of reactive oxygen species generated. This suggests that brain MDMA concentration is the determinant in the injury initiation while reactive oxygen species generation associated with the injury intensification. It is concluded that the two adverse events utilize distinctly different mediating molecules during the toxic initiation and intensification.

The Mediating Role of A2A Adenosine Receptors in the Mitochondrial Pathway of Apoptotic Hippocampal Cell Death, Following the Administration of MDMA in Rat.

Introduction:The 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) is a popular recreational drug and a major source of substance abuse, which ultimately leads to sensations of well-being, elation and euphoria, moderate derealization/depersonalization, and cognitive disruptions, as well as intense sensory awareness. The mechanisms involved in memory impairment induced by MDMA are not completely understood.Methods:The current study used 40 Sprague-Dawley rats, weighted 200 to 250 g. Experiments were performed in four groups, each containing 10 rats. The first group of rats was used as the control, treated with dimethyl sulfoxide (DMSO). The second group was treated with MDMA. The third group was treated with MDMA and CGS (the adenosine A2A receptor agonist, 2-[p-(2-carboxyethyl) phenethylamino]-5′-N-ethylcarboxamidoadenosine) (CGS 21680) and the fourth group was treated with MDMA and SCH (the A2A receptor antagonist [7-(2-phenylethyl)-5-amino-2-(2-furyl-) pyrazolo-[4, 3-e]-1, 2, 4 triazolo [1,5-] pyrimidine]) (SCH 58261). The drugs in all groups were administrated intraperitoneally (i.p.) once a day for 7 days. In 5 rats of each group, following perfusion, samples were taken from hippocampi to investigate apoptosis. Accordingly, the samples were stained using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay kit, and studied by light microscopy. In other rats, fresh tissue was also removed to study the expression of bax and bcl-2 by Western blotting technique.Results:It was observed that the coadministration of MDMA with CGS reduced bax expression and prevented apoptosis of hippocampal cells. The coadministration of MDMA and SCH increased bax expression, and also increased the frequency of hippocampal cell apoptosis.Conclusion:The results of the current study showed that administration of CGS with MDMA decreased the common side effects associated with MDMA.

Sex differences in abuse-related neurochemical and behavioral effects of 3,4-methylenedioxymethamphetamine (MDMA) in rats

3,4-Methylenedioxymethamphetamine (MDMA) is a substrate for dopamine (DA), norepinephrine and serotonin (5HT) transporters that produces greater pharmacological effects on certain endpoints in females than males in both clinical and rodent preclinical studies. To evaluate potential for sex differences in abuse-related MDMA effects, the present study compared MDMA effects on intracranial self-stimulation (ICSS) and on in vivo microdialysis measurements of DA or 5HT in the nucleus accumbens (NAc) in female and male Sprague-Dawley rats. For ICSS studies, electrodes were implanted in the medial forebrain bundle and rats trained to press for electrical stimulation over a range of frequencies (56–158Hz, 0.05 log increments) under a fixed-ratio 1 schedule, and the potency (0.32–3.2mg/kg, 10min pretreatment) and time course (3.2. mg/kg, 10–180min pretreatment) of MDMA effects were determined. For in vivo microdialysis, rats were implanted with bilateral guide cannulae targeting the NAc, and the time course of MDMA effects (1.0–3.2mg/kg, 0–180min) on DA and 5HT was determined. MDMA produced qualitatively similar effects in both sexes on ICSS (both increases in low ICSS rates maintained by low brain-stimulation frequencies and decreases in high ICSS rates maintained by high brain-stimulation frequencies) and microdialysis (increases in both DA and 5HT). The duration and peak levels of both abuse-related ICSS facilitation and increases in NAc DA were longer in females. MDMA was also more potent to increase 5HT in females. These results provide evidence for heightened sensitivity of females to abuse-related behavioral and neurochemical effects of MDMA in rats.

Caffeine alters the behavioural and body temperature responses to mephedrone without causing long-term neurotoxicity in rats.

Administration of caffeine with 3,4-methylenedioxymethamphetamine (MDMA) alters the pharmacological properties of MDMA in rats. The current study examined whether caffeine alters the behavioural and neurochemical effects of mephedrone, which has similar psychoactive effects to MDMA. Rats received either saline, mephedrone (10 mg/kg), caffeine (10 mg/kg) or combined caffeine and mephedrone intraperitoneally twice weekly on consecutive days for three weeks. Locomotor activity (days 1 and 16), novel object discrimination (NOD, day 2), elevated plus maze (EPM) exploration (day 8), rectal temperature changes (day 9) and pre-pulse inhibition (PPI) of acoustic startle response (day 15) were assessed. Seven days after the final injection, brain regions were collected for the measurement of 5-hydroxytryptamine (5-HT), dopamine and their metabolites. Combined caffeine and mephedrone further enhanced the locomotor response observed following either drug administered alone, and converted mephedrone-induced hypothermia to hyperthermia. Co-administration also abolished mephedrone-induced anxiogenic response on the EPM, but had no effect on NOD or PPI. Importantly, no long-term neurotoxicity was detected following repeated mephedrone alone or when co-administered with caffeine. In conclusion, the study suggests a potentially dangerous effect of concomitant caffeine and mephedrone, and highlights the importance of taking polydrug use into consideration when investigating the acute adverse effect profile of popular recreational drugs.

Pentoxifylline Protects the Rat Liver Against Fibrosis and Apoptosis Induced by Acute Administration of 3,4-Methylenedioxymethamphetamine (MDMA or Ecstasy).

3,4-Methylenedioxymethamphetamine (MDMA) is one of the most popular drugs of abuse in the world with hallucinogenic properties that has been shown to induce apoptosis in liver cells. The present study aimed to investigate the effects of pentoxifylline (PTX) on liver damage induced by acute administration of MDMA in Wistar rat. Animals were administered with saline or MDMA (7.5 mg/kg, IP) 3 times with 2 hr intervals. PTX (200 mg kg, IP), was administered simultaneously with last injection of MDMA in experimental group. The concomitant administration of pentoxifylline and MDMA decreased liver injury including apoptosis, fibrosis and hepatocytes damages. Our results showed for the first time that PTX treatment diminishes the extent of apoptosis and fibrosis caused by MDMA in rat liver.

A genetic deletion of the serotonin transporter greatly enhances the reinforcing properties of MDMA in rats

A genetic deletion of the serotonin transporter greatly enhances the reinforcing properties of MDMA in rats

3,4-Methylenedioxy-methamphetamine induces in vivo regional up-regulation of central nicotinic receptors in rats and potentiates the regulatory effects of nicotine on these receptors

Nicotine (NIC), the main psychostimulant compound of smoked tobacco, exerts its effects through activation of central nicotinic acetylcholine receptors (nAChR), which become up-regulated after chronic administration. Recent work has demonstrated that the recreational drug 3,4-methylenedioxy-methamphetamine (MDMA) has affinity for nAChR and also induces up-regulation of nAChR in PC 12 cells. Tobacco and MDMA are often consumed together. In the present work we studied the in vivo effect of a classic chronic dosing schedule of MDMA in rats, alone or combined with a chronic schedule of NIC, on the density of nAChR and on serotonin reuptake transporters. MDMA induced significant decreases in [3H]paroxetine binding in the cortex and hippocampus measured 24h after the last dose and these decreases were not modified by the association with NIC. In the prefrontal cortex, NIC and MDMA each induced significant increases in [3H]epibatidine binding (29.5 and 34.6%, respectively) with respect to saline-treated rats, and these increases were significantly potentiated (up to 72.1%) when the two drugs were associated. Also in this area, [3H]methyllycaconitine binding was increased a 42.1% with NIC+MDMA but not when they were given alone. In the hippocampus, MDMA potentiated the α7 regulatory effects of NIC (raising a 25.5% increase to 52.5%) but alone was devoid of effect. MDMA had no effect on heteromeric nAChR in striatum and a coronal section of the midbrain containing superior colliculi, geniculate nuclei, substantia nigra and ventral tegmental area. Specific immunoprecipitation of solubilised receptors suggests that the up-regulated heteromeric nAChRs contain α4 and β2 subunits. Western blots with specific α4 and α7 antibodies showed no significant differences between the groups, indicating that, as reported for nicotine, up-regulation caused by MDMA is due to post-translational events rather than increased receptor synthesis.

Caffeine provokes adverse interactions with 3,4-methylenedioxymethamphetamine (MDMA, 'ecstasy') and related psychostimulants: mechanisms and mediators.

Concomitant consumption of caffeine with recreational psychostimulant drugs of abuse can provoke severe acute adverse reactions in addition to longer term consequences. The mechanisms by which caffeine increases the toxicity of psychostimulants include changes in body temperature regulation, cardiotoxicity and lowering of the seizure threshold. Caffeine also influences the stimulatory, discriminative and reinforcing effects of psychostimulant drugs. In this review, we consider our current understanding of such caffeine-related drug interactions, placing a particular emphasis on an adverse interaction between caffeine and the substituted amphetamine, 3,4-methylenedioxymethamphetamine (MDMA, 'ecstasy'), which has been most recently described and characterized. Co-administration of caffeine profoundly enhances the acute toxicity of MDMA in rats, as manifested by high core body temperature, tachycardia and increased mortality. In addition, co-administration of caffeine enhances the long-term serotonergic neurotoxicity induced by MDMA. Observations to date support an interactive model of drug-induced toxicity comprising MDMA-related enhancement of dopamine release coupled to a caffeine-mediated antagonism of adenosine receptors in addition to inhibition of PDE. These experiments are reviewed together with reports of caffeine-related drug interactions with cocaine, d-amphetamine and ephedrine where similar mechanisms are implicated. Understanding the underlying mechanisms will guide appropriate intervention strategies for the management of severe reactions and potential for increased drug-related toxicity, resulting from concomitant caffeine consumption.

Behavioural and neurochemical comparison of chronic intermittent cathinone, mephedrone and MDMA administration to the rat

The synthetic cathinone derivative, mephedrone, is a controlled substance across Europe. Its effects have been compared by users to 3,4-methylenedioxymethamphetamine (MDMA), but little data exist on its pharmacological properties. This study compared the behavioural and neurochemical effects of mephedrone with cathinone and MDMA in rats. Young-adult male Lister hooded rats received i.p. cathinone (1 or 4mg/kg), mephedrone (1, 4 or 10mg/kg) or MDMA (10mg/kg) on two consecutive days weekly for 3 weeks or as a single acute injection (for neurochemical analysis). Locomotor activity (LMA), novel object discrimination (NOD), conditioned emotional response (CER) and prepulse inhibition of the acoustic startle response (PPI) were measured following intermittent drug administration. Dopamine, 5-hydroxytryptamine (5-HT) and their major metabolites were measured in striatum, frontal cortex and hippocampus by high performance liquid chromatography 7 days after intermittent dosing and 2h after acute injection. Cathinone (1, 4mg/kg), mephedrone (10mg/kg) and MDMA (10mg/kg) induced hyperactivity following the first and sixth injections and sensitization to cathinone and mephedrone occurred with chronic dosing. All drugs impaired NOD and mephedrone (10mg/kg) reduced freezing in response to contextual re-exposure during the CER retention trial. Acute MDMA reduced hippocampal 5-HT and 5-HIAA but the only significant effect on dopamine, 5-HT and their metabolites following chronic dosing was altered hippocampal 3,4-dihydroxyphenylacetic acid (DOPAC), following mephedrone (4, 10mg/kg) and MDMA. At the doses examined, mephedrone, cathinone, and MDMA induced similar effects on behaviour and failed to induce neurotoxic damage when administered intermittently over 3 weeks.

Ecstasy cannot be assumed to be 3,4‐methylenedioxyamphetamine (MDMA)

LINKED ARTICLESThis is a rebuttal by the authors (Green et al., pp. 1523–1536 of this issue) to a commentary by Parrott, pp. 1518–1520 of this issue. To view the article by Green et al. visit http://dx.doi.org/10.1111/j.1476‐5381.2011.01819.x. To view the commentary by Parrott visit http://dx.doi.org/10.1111/j.1476‐5381.2012.01941.x

Pro‐social effects of 3,4‐methylenedioxymethamphetamine in mice

The drug 3,4‐methylenedioxymethamphetamine (MDMA, “ecstasy”) has been shown to increase feelings of closeness and empathy in humans. Despite evidence of MDMA‐induced neurotoxicity, these unusual pro‐social effects have continued to generate interest in the drug's therapeutic potential. Therefore, determining the mechanisms by which MDMA engenders these effects may allow for the development of novel, pro‐social medications that do not exhibit neurotoxicity. A number of genetic and molecular techniques that may be critical for understanding these mechanisms are primarily available in mice, yet the effects of MDMA on murine social behavior have not previously been described. To this end, in the present study, we characterized the effects of (+/−)‐MDMA in mice across a range of doses (3–30mg/kg, IP) on dyadic social behavior when mice in a pair were treated with the same dose of MDMA. We found that MDMA increased the duration of time the pair spent in close proximity, an effect that is consistent with the previously reported effects of MDMA in rats. In subsequent experiments, we will use selective receptor antagonists to further elucidate the mechanisms underlying this effect. Understanding these mechanisms may provide insight into the etiology of disorders exhibiting social deficits and help identify targets for their treatment. This study was supported with funds from the USPHS: DA020645 and RR00165.

Effects of MDMA (ecstasy) and two of its metabolites on rat embryos in vitro

MDMA consumers are young people of childbearing age. Consequently, developmental exposure to this drug is a potential public health concern. Several studies have addressed MDMA neurotoxicity in adults; however, knowledge of the effects of MDMA on developing embryos is limited. After administration, MDMA is metabolized species specifically via two main pathways. One leads to the formation of MDA and the other to the formation of HHMA. Here we evaluated the embryotoxic effects of MDMA, and also those of MDA, a main metabolite of MDMA in rats, and HHMA, a main metabolite in humans. For this purpose, we used the whole embryo culture (WEC). Our results show a concentration-dependent embryotoxic effect of MDMA, MDA and HHMA at a concentration range of 25–50μg/ml. The embryotoxic potential of the parent compound and the two metabolites was comparable in vitro.

Evaluating the Contribution of Serotonin Receptor Subtypes and ‘Binge’ 3,4-Methylenedioxymethamphetamine (MDMA) Exposure to the Discriminative Stimulus Effects of MDMA in Rats

3,4-Methylenedioxymethamphetamine (MDMA; ‘Ecstasy’) shares psychoactive effects with drugs that possess stimulant (e.g. amphetamine, the effects of which are primarily dopaminergic) and hallucinogenic properties (e.g. LSD, which has serotonergic effects) [1]. The majority of MDMA’s distinctive effects as well as its toxicity have been linked to its actions on serotonergic neurotransmission [2]. One way in which MDMA’s serotonergic effects can be studied is to train rats to distinguish dopaminergic stimulant effects from mood and perception-altering serotonergic effects using a three-way drug discrimination paradigm [3].

The role of monoamines in the changes in body temperature induced by 3,4‐methylenedioxymethamphetamine (MDMA, ecstasy) and its derivatives

Hyperthermia is probably the most widely known acute adverse event that can follow ingestion of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) by recreational users. The effect of MDMA on body temperature is complex because the drug has actions on all three major monoamine neurotransmitters [5-hydroxytryptamine (5-HT), dopamine and noradrenaline], both by amine release and by direct receptor activation. Hyperthermia and hypothermia can be induced in laboratory animals by MDMA, depending on the ambient temperature, and involve both central thermoregulation and peripheral changes in blood flow and thermogenesis. Acute 5-HT release is not directly responsible for hyperthermia, but 5-HT receptors are involved in modulating the hyperthermic response. Impairing 5-HT function with a neurotoxic dose of MDMA or p-chlorophenylalanine alters the subsequent MDMA-induced hyperthermic response. MDMA also releases dopamine, and evidence suggests that this transmitter is involved in both the hyperthermic and hypothermic effects of MDMA in rats. The noradrenergic system is also involved in the hyperthermic response to MDMA. MDMA activates central alpha(2A)-adrenoceptors and peripheral alpha(1)-adrenoceptors to produce cutaneous vasoconstriction to restrict heat loss, and beta(3)-adrenoceptors in brown adipose tissue to increase heat generation. The hyperthermia occurring in recreational users of MDMA can be fatal, but data reviewed here indicate that it is unlikely that any single pharmaceutical agent will be effective in reversing the hyperthermia, so careful body cooling remains the principal clinical approach. Crucially, educating recreational users about the potential dangers of hyperthermia and the control of ambient temperature should remain key approaches to prevent this potentially fatal problem.

Drug seeking in response to a priming injection of MDMA in rats: relationship to initial sensitivity to self-administered MDMA and dorsal striatal dopamine

In laboratory animals, exposure to priming injections of 3,4-methylenedioxymethamphetamine (MDMA) produced drug seeking following extinction of MDMA self-administration. This study aimed to evaluate whether the magnitude of drug seeking was related to latency to acquisition of MDMA self-administration and increases in striatal dopamine, as measured by in-vivo microdialysis. Rats were given daily access to MDMA self-administration until they earned a total of 240 infusions (total intake of 165 mg/kg MDMA). Twelve of the 20 rats acquired self-administration within the temporal limits of the study and the latency to meet the criterion ranged from 9 d to 37 d. An experimenter-administered injection of MDMA (10.0 mg/kg i.p.) produced drug seeking in these rats, and the number of responses was significantly higher than responses produced by rats that failed to meet the criterion or by yoked control rats that received the drug passively. For rats that met the criterion, drug seeking was negatively correlated with the number of days to self-administer the criterion number of MDMA infusions and positively correlated with MDMA-produced dopamine in the dorsal striatum. Importantly, MDMA-produced dopamine overflow was greater for the rats that met the criterion. These findings suggest that drug seeking is influenced by initial sensitivity to the reinforcing effects of MDMA and to drug-produced increases in striatal dopamine.

Determination of a new designer drug, N-hydroxy-3,4-methylenedioxymethamphetamine and its metabolites in rats using ultra-performance liquid chromatography–tandem mass spectrometry

An N-hydroxy analogue of 3,4-methylendioxymethamphetamine (MDMA), N-hydroxy MDMA ( N-OH MDMA), has recently been distributed as a new designer drug in some drug markets. Very little data is available to the metabolic and pharmacological properties of N-OH MDMA, although it has been reported that the N-demethyl analogue, N-hydroxy-3,4-methylenedioxyamphetamine ( N-OH MDA), is mainly metabolized to MDA in rats. In this study, an analytical method for the determination of N-OH MDMA and its metabolites in biological samples was developed, and the metabolic properties of N-OH MDMA in rats were investigated. After the i.p. administration of N-OH MDMA to pigmented hairy rats (5 mg/kg/day, 10 days), N-OH MDMA and its N-dehydroxy and N-demethyl metabolites (MDMA, N-OH MDA and MDA) in rat plasma, urine and hair samples were determined by ultra-performance LC (UPLC)–MS/MS. The hair sample was extracted by 1-h sonication and overnight soaking in 5 M hydrochloric acid–methanol (1:20). The plasma, urine, and hair extract samples were purified using a solid-phase extraction procedure. N-OH MDMA in the samples could be precisely analyzed by avoiding an alkaline environment. The parent compound very rapidly disappeared from the rat plasma (<15 min) and urine (<10 h), and most of the N-OH MDMA was excreted in the rat urine as MDMA and MDA in 72 h. In the rat hair samples collected 4 weeks after the first administration, N-OH MDMA (0.03 ng/mg) and N-OH MDA (0.13 ng/mg) were clearly detected as well as MDMA (149 ng/mg) and MDA (52 ng/mg). This analytical method will be useful for the analysis of N-OH MDMA and its metabolites in biological samples.