Development and validation of a disk solid phase extraction and gas chromatography-mass spectrometry method for MDMA, MDA, HMMA, HMA, MDEA, methamphetamine and amphetamine in sweat

We describe the development and validation of a method for the simultaneous quantification of 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), 3-hydroxy-4-methoxymethamphetamine (HMMA), 3-hydroxy-4-methoxyamphetamine (HMA), 3,4-methylenedioxyethylamphetamine (MDEA), methamphetamine (MAMP) and amphetamine (AMP) in sweat. Drugs were eluted from PharmChek sweat patches with sodium acetate buffer, extracted with disk solid phase extraction and analyzed using GC/MS-EI with selected ion monitoring. Limits of quantification (LOQ) for MDMA, MDEA, MAMP and AMP were 2.5 ng/patch, and 5 ng/patch for MDA, HMA and HMMA. This fully validated procedure was more sensitive than previously published analytical methods and permitted the simultaneous analysis of multiple amphetamine analogs in human sweat.     

Stereochemistry of the metabolism of MDMA to MDA

The chiral derivatizing reagent N-trifluoroacetyl-L-prolyl chloride (LTPC) was used to form diastereomers of 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA) which were resolved on an achiral gas chromatographic column using a mass spectrometer as a detector. Rats were subcutaneously dosed with 40 mg/kg of (+/-) MDMA.HCl and blood was obtained by decapitation four hours after dosing. Plasma was separated and extracted. The extract was derivatized on-column with LTPC. In addition to the two MDMA isomers, the demethylated metabolites, S(+) and R(-)-MDA were identified. In all experimental groups (male rats, food deprived male rats, female rats, post partum female rats, and mice) dosed with racemic MDMA, higher levels of the S(+) isomer of MDA relative to the R(-) MDA isomer were observed. This may be significant since it has been shown that the S(+) isomer of MDMA is the more neurotoxic isomer of the racemic drug of abuse MDMA.     

Gas chromatography–ion trap mass spectrometry method for the simultaneous measurement of MDMA (ecstasy) and its metabolites,MDA, HMA,and HMMA in plasma and urine

The investigation of 3,4-methylenedioxymethamphetamine (MDMA; ecstasy) abuse requires very robust methods with high sensitivity and wide linearity ranges for the quantification of this drug of abuse and its main metabolites in body fluids. An optimized gas chromatography–ion trap mass spectrometry (GC–IT/MS) methodology with electron impact ionization addressing these issues is presented. The sample preparation involves an enzymatic hydrolysis of urine and plasma for conjugate cleavage, a SPE extraction, and a derivatization process. The method was fully validated in rat plasma and urine. Linearity for a wide concentration range was achieved for MDMA, and the metabolites 3,4-methylenedioxyamphetamine (MDA), 4-hydroxy-3-methoxyamphetamine (HMA) and 4-hydroxy-3-methoxymethamphetamine (HMMA). Limits of quantification were 2 ng/mL in plasma and 3.5 ng/mL in urine using a Selected Ion Monitoring detection mode. Selectivity, accuracy, precision, and recovery met the required criteria for the method validation. This GC–IT/MS method provides high sensitivity and adequate performance characteristics for the simultaneous quantification of MDMA, MDA, HMA and HMMA in the studied matrices.     

Pentazocine monitoring in rat hair and plasma by HPLC-fluorescence detection with DIB-Cl as a labelling reagent.

Pentazocine (PZ) in rat hair and plasma was determined by HPLC-fluorescence detection with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) as a labelling reagent and cyclazocine (CZ) as an internal standard (IS). PZ and IS extracted from hair or plasma sample were derivatized with DIB-Cl and the resulted solution was cleaned up with solid phase extraction. The isocratic separation of DIB-PZ and -CZ within 20 min could be achieved by a Wakopak Handy-ODS column (250 x 4.6 mm i.d.) using a mobile phase composed of 0.1 mol/L acetate buffer (pH 6.2):acetonitrile (25:75, v/v). The detection limits of PZ at a signal-to-noise ratio of 3 for rat hair and plasma were 0.18 ng/mg and 0.57 ng/mL, respectively. Reproducible and precise results could be obtained by an IS method with RSD values less than 6.6% for within- and between-day measurements. The method was successfully applied for the monitoring of PZ levels in Zucker rat hair and plasma samples after a single administration of 25 mg/kg PZ. Moreover, incorporation rates of PZ into black and white hair of Zucker rat were evaluated.     

Determination of erythromycin in rat plasma with capillary electrophoresis-electrochemiluminescence detection of tris(2,2'-bipyridyl) ruthenium(II)

A fast and sensitive approach for determination of erythromycin in rat plasma was described. The method used capillary electrophoresis coupled with end-column electrochemiluminescence (ECL) detection of Ru(bpy)(3)(2+). The separation column used had an inner diameter of 75 microm. The running buffer was 15 mmol/L sodium phosphate (pH=7.5). The solution in the detection cell was 50 mmol/L sodium phosphate (pH=8.0) and 5 mmol/L Ru(bpy)(3)(2+). ECL intensity varied linearly with erythromycin concentration from 1.0 ng/mL to 10 microg/mL. The detection limit (S/N=3) was 0.35 ng/mL. The relative standard deviations, of ECL intensity and migration time for eight consecutive injections of 1.0 microg/mL erythromycin (n=8), were 1.3% and 1.8%, respectively. The method was successfully applied to erythromycin determination in rat plasma. The recovery ranged from 92.5 to 97.5%.     

Simultaneous determination of the major active components of tea polyphenols in rat plasma by a simple and specific HPLC assay

A simple and specific HPLC assay for simultaneous determination of two major active components (-) epigallocatechin-3-gallate (EGCG), and (-) epicatechin-3-gallate (ECG) of tea polyphenols (TP) in rat plasma was developed and validated. Following addition of resorcinol as internal standard (IS) the analytes were isolated from rat plasma by liquid-liquid extraction with ethyl acetate. The chromatographic separation was achieved on a reversed-phase C18 column using an isocratic mobile phase consisting of 0.1% citric acid+CH(3)CN (86:14, v/v) running at flow rate of 1.5 mL/min. The effluent was monitored at a wavelength of 280 nm. EGCG, ECG and IS were well separated from each other and free from interference from blank plasma and other components in TP as well as metabolites post-dosing. The calibration curve was constructed by plotting peak area ratio of analytes to IS vs. concentration. The method showed good linearity over range of 0.5-300 microg/mL for EGCG and 0.1-60 microg/mL for ECG (r>0.999). The intra- and inter-day precision (R.S.D.) was better than 6 and 12%, respectively. Assay accuracy was better than 94.78% for both compounds. Extraction recovery at QC samples was between 85.73 and 91.93% for EGCG and 79.08 and 86.51% for ECG. The developed method was successfully used to simultaneously measure plasma concentrations of EGCG and ECG after intravenous administration of TP to rats and yielded two typical biexponential decay concentration-time curves.     

Confirmation of amphetamine,methamphetamine, MDA and MDMA in urine samples using disk solid-phase extraction and gas chromatography-mass spectrometry after immunoassay screening

A method using mixed phase disk solid-phase extraction (SPE) and gas chromatography-mass spectrometry (GC-MS) was developed for confirmation of amphetamine (AMP), methamphetamine (MET), 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethamphetamine (MDMA) in urine samples after immunoassay screening. Disk SPE provided hydrophobic (C(18)) and strong cation-exchange (SCX) interactions. The analytes were retained on SCX functional groups in the disk and eluted with ammoniated ethyl acetate after washed with methanol. Confirmation and quantitation was exercised by selected ion monitoring using nikethamide as chromatographic standard. Recoveries of the amphetamines were between 73.0 and 104.6% with RSDs in range of 2.1-6.4% (n=3). The limits of detection were 2 ng/ml for AMP, MET and MDMA, and 4 ng/ml for MDA. Five real urine samples were tested with the method after immunoassay screening, and the results were comparable to those of traditional liquid-liquid extraction (LLE). The method was solvent-saved, simple, rapid and reliable, and the extract was cleaner than that of LLE.     

Development and validation of a LC-ESI-MS/MS method for the determination of swertiamarin in rat plasma and its application in pharmacokinetics

A new LC-ESI-MS/MS assay method has been developed and validated for the quantification of swertiamarin, a representative bioactive substance of Swertia plants, in rat plasma using gentiopicroside, an analog of swertiamarin on chemical structure and chromatographic action, as the internal standard (IS). The swertiamarin and IS were extracted from rat plasma using solid-phase extraction (SPE) as the sample clean-up procedure, and they were chromatographed on a narrow internal diameter column (Agilent ZORBAX ECLIPSE XDB-C(18) 100 mm × 2.1 mm, 1.8 μm) with the mobile phase consisting of methanol and water containing 0.1% acetic acid (25:75, v/v) at a flow rate of 0.2 mL/min. The detection was performed on an Agilent G6410B tandem mass spectrometer by negative ion electrospray ionisation in multiple-reaction monitoring mode while monitoring the transitions of m/z 433 [M+CH(3)COO](-)→179 and m/z 415 [M+CH(3)COO](-)→179 for swertiamarin and IS, respectively. The lower limit of quantification (LLOQ) was 5 ng/mL within a linear range of 5-1000 ng/mL (n=7, r(2)≥0.994), and the limit of detection (LOD) was demonstrated as 1.25 ng/mL (S/N≥3). The method also afforded satisfactory results in terms of sensitivity, specificity, precision (intra- and inter-day), accuracy, recovery, freeze/thaw, long-time stability and dilution integrity. This method was successfully applied to determination of the pharmacokinetic properties of swertiamarin in rats after oral administration at a dose of 20 mg/kg. The following pharmacokinetic parameters were obtained (mean): maximum plasma concentration, 1920.1 ng/mL; time to reach maximum plasma concentration, 0.945 h; elimination half-time, 1.10h; apparent total clearance, 5.638 L/h/kg; and apparent volume of distribution, 9.637 L/kg.     

Determination of 3,4-methylenedioxymethamphetamine and its five main metabolites in rat urine by solid-phase extraction and high performance liquid chromatography with on line mass spectrometry

The consumption of psychostimulant amphetamine-like drugs has increased significantly in recent years. Some MDMA metabolites are probably involved in the neurotoxicity and neurodegeneration caused by prolonged use rather than MDMA itself. We recently developed a method to analyze MDMA and its five main metabolites in rat plasma [7]. We have now fully validated this method to the quantification of these drugs in rat urine. We extracted MDMA and its metabolites with Oasis WCX cartridges, separated them on a Nucleodur C18 analytical column and quantified them by ion-trap mass spectrometry. Linearity was excellent: 12.5–1250 ng/mL urine for HMA, HMMA, MDA and MDMA, 25–2500 ng/mL for HHMA, and 150–7500 ng/mL for HHA (r² > 0.993 for all analytes). The lower limits of quantification were 12.5 ng/mL urine for MDMA, MDA, HMA and HMMA, 25 ng/mL for HHMA and 150 ng/mL for HHA. Reproducibility was good (intra-assay precision = 1.7–6.1%; inter-assay precision = 0.6–5.7%), as was accuracy (intra-assay deviation = 0.1–4.8%; inter-assay deviation = 0.7–7.9%). Average recoveries were around 85.0%, except for HHMA (66.2%) and HHA (53.0%) (CV < 8.3%). We also checked the stability of stock solutions and the internal standards after freeze-thawing and in the autosampler. Lastly, we measured the MDMA, MDA, HHMA, HHA, HMMA and HMA in urine samples taken over 24 h from rats given subcutaneous MDMA.     

High-performance liquid chromatography with electrochemical detection applied to the analysis of 3,4-dihydroxymethamphetamine in human plasma and urine

Metabolic activation in the disposition of 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") has been implicated in some of its pharmacological and toxicological effects, with the major metabolite 3,4-dihydroxymethamphetamine (HHMA) as a putative toxicant through the formation of thioether adducts. We describe the first validated method for HHMA determination based on acid hydrolysis of plasma and urine samples, further extraction by a solid-phase strong cation-exchange resin (SCX, benzenesulfonic acid), and analysis of extracts by high-performance liquid chromatography with electrochemical detection. The chromatographic separation was performed in an n-butyl-silane (C4) column and the mobile phase was a mixture of 0.1 M sodium acetate containing 0.1 M 1-octanesulphonic acid and 4 mM EDTA (pH 3.1) and acetonitrile (82:18, v/v). Compounds were monitored with an electrochemical cell (working potentials 1 and 2, +0.05 and +0.35 V, respectively, gain 60 microA). A mobile phase conditioning cell with a potential set at +0.40 V was connected between the pumping system and the injector. Calibration curves were linear within the working concentration ranges of 50-1000 microg/L for urine and plasma. Limits of detection and quantification were 10.5 and 31.8 microg/L for urine and 9.2 and 28.2 microg/L for plasma. Recoveries for HHMA and DHBA (3,4-dihydroxybenzylamine, internal standard) were close to 50% for both biological matrices. Intermediate precision and inter-day accuracy were within 3.9-6.5% and 7.4-15.3% for urine and 5.0-10.8% and 9.2-13.4% for plasma.     

Quantification of 3,4-methylenedioxymetamphetamine and its metabolites in plasma and urine by gas chromatography with nitrogen–phosphorus detection

A gas chromatographic method with nitrogen–phosphorus detection involving a solid–liquid extraction phase was developed and validated for the simultaneous quantification of 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA) in plasma. A modification of this method was validated for the analysis of MDMA, MDA, 4-hydroxy-3-methoxymethamphetamine (HMMA) and, 4-hydroxy-3-methoxyamphetamine (HMA) in urine. Under the analytical conditions described, the limits of detection in plasma and urine were less than 1.6 μg/l and 47 μg/l, respectively, for all the compounds studied. Good linearity was observed in the concentration range evaluated in plasma (5–400 μg/l) and urine (100–2000 μg/l) for all compounds tested. The recoveries obtained from plasma were 85.1% and 91.6% for MDMA and MDA, respectively. Urine recoveries were higher than 90% for MDMA and MDA, 74% for HMMA, and 64% for HMA. Methods have been successfully used in the assessment of plasma and urine concentrations of MDMA and its main metabolites in samples from clinical studies in healthy volunteers.     

Direct determination of glucuronide and sulfate of 4-hydroxy-3-methoxymethamphetamine, the main metabolite of MDMA, in human urine

A sensitive and reliable LC-ESI-MS procedure for the simultaneous determination of MDMA and its five metabolites including 4-hydroxy-3-methoxymethamphetamine (HMMA) conjugates has been established following the synthesis of two HMMA conjugates, 4-hydroxy-3-methoxymethamphetamine-glucuronide (HMMA-Glu) and 4-hydroxy-3-methoxymethamphetamine-sulfate (HMMA-Sul). Pretreatment of urine samples with methanol and LC-MS employing a C(18) semi-micro column with a gradient elution program provided the successful separations and MS determinations of these analytes within 20 min. Upon applying the method to MDMA users' urine specimens, HMMA-Glu and HMMA-Sul have been directly determined, suggesting the superiority of sulfation to glucuronidation in the HMMA phase II metabolism.     

A validated gas chromatographic-electron impact ionization mass spectrometric method for methylenedioxymethamphetamine (MDMA), methamphetamine and metabolites in oral fluid

An analytical method to simultaneously quantify amphetamine (AMP), methamphetamine (MAMP), methylenedioxymethamphetamine (MDMA), methylenedioxyamphetamine (MDA), methylenedioxyethylamphetamine (MDEA), 3-hydroxy-4-methoxy-methamphetamine (HMMA) and 3-hydroxy-4-methoxy-amphetamine (HMA) in oral fluid is presented. Four hundred microlitres of oral fluid collected via expectoration was extracted by solid phase extraction. GC/MS-EI with selected ion monitoring (SIM) yielded linear curves 5-250 ng/mL for AMP, MAMP, MDMA and MDEA, 5-500 ng/mL for MDA and 25-1,000 ng/mL for HMA and HMMA. Recoveries were greater than 85%, accuracy 87-104%, and precision less than 8.3% coefficient of variation. This assay will be used to investigate distribution of sympathomimetic amines into human oral fluid following controlled drug administration.     

HPLC-UV method development for fentanyl determination in rat plasma and its application to elucidate pharmacokinetic behavior after i.p. administration to rats

A simple, rapid and validated high performance liquid chromatography method with UV detection for the quantification of an opioid agonist, fentanyl (FEN), in rat plasma was developed. The assay procedure involved chromatographic separation using a ZIC-HILIC SeQUANT column (250 mm × 4.6 mm, i.d., 5 μm) and a mobile phase of acetonitrile and acetate buffer (pH 3.4, 20mM) of ratio (=65:35, v/v) at a flow rate of 1.2 mL/min and detection wavelength of 201 nm. Plasma sample (100 μL) pretreatment was based on simple deprotienization by acetonitrile spiked with clonidine as an internal standard (I.S.) of 20 ng/mL followed by extraction with tert-butyl methyl ether and centrifugation. The organic layer was evaporated under N(2) gas and reconstituted with 100 μL of acetate buffer (pH 3.4, 20mM), and 50-μL portions of reconstituted sample were injected onto the column. Sample analysis including sample pretreatment was achieved within 35 min. Calibration curve was linear (r ≥ 0.998) from 5 to 100 ng/mL. Both intra- and inter-day assay precisions that are presented through RSD were lower than 12.6% for intra-day and lower than 12.0% for inter-day assessment. Limit of detection was 0.8 ng/mL at S/N of 3. This method was omitting the use of expensive solid phase extraction and time consuming liquid extraction procedures. Moreover, the present method was successfully applied to study pharmacokinetic parameters of FEN after intraperitoneal administration to male Wistar rat. Pharmacokinetic parameters estimated by using moment analysis were T(1/2) 198.3 ± 44.7 min, T(max) 28.3 ± 2.9 min and AUC(0-180) 15.6 ± 2.9(× 10(2))ngmin/mL.     

Development and validation of an HPLC method for the determination of dibenzoylmethane in rat plasma and its application to the pharmacokinetic study

A highly sensitive and simple high-performance liquid chromatographic (HPLC) assay has been developed and validated for the quantification of dibenzoylmethane (DBM) in rat plasma. DBM and internal standard (I.S.) 1-(5-chloro-2-hydroxy-4-methylphenyl)-3-phenyl-1,3-propanedione (CHMPP) were extracted from rat plasma by ethyl acetate/methanol (95:5, v/v) and analyzed using reverse-phase gradient elution with a Phenomenex Gemini C18 5-mum column. A gradient of mobile phase (mobile phase A: water/methanol (80:20, v/v) with 0.1% TFA and mobile phase B: acetonitrile with 0.1% TFA) at a flow rate of 0.2 mL/min, and ultraviolet (UV) detection at 335 nm were utilized. The lower limit of quantification (LLOQ) using 50 microL rat plasma was 0.05 microg/mL. The calibration curve was linear over a concentration range of 0.05-20 microg/mL. The mean recoveries were 80.6+/-5.7, 83.4+/-1.6 and 77.1+/-3.4% with quality control (QC) level of 0.05, 1 and 20 microg/mL, respectively. Intra- and inter-day assay accuracy and precision fulfilled US FDA guidance for industry bioanalytical method validation. Stability studies showed that DBM was stable in rat plasma after 4h incubation at room temperature, one month storage at -80 degrees C and three freeze/thaw cycles, as well as in reconstitute buffer for 48 h at 4 degrees C. The utility of the assay was confirmed by the successful analysis of plasma samples from DBM pharmacokinetics studies in the rats after oral and intravenous administrations.     

Development and validation of an LC-MS method with electrospray ionization for quantitation of digoxin in human plasma and urine: application to a pharmacokinetic study

A highly sensitive and specific LC-MS method was developed and validated for the quantification of digoxin in human plasma and urine using d5-dihydrodigoxin as internal standard (IS). The assay procedure involved extraction of digoxin and IS from human plasma with chloroform-isopropanol (95:5, v/v). Chromatogrphic separation was achieved on a Spherisorb ODS2 column using a gradient mobile phase with 5 mmol/L ammonium acetate in water with 1% acetic acid and acetonitrile. The mass spectrometer was operated in the selected ion monitoring mode using the respective [M+K](+) ions, m/z 819.4 for digoxin and m/z 826.4 for IS. The method was proved to be accurate and precise at linearity range of 0.12-19.60 ng/mL in plasma with a correlation coefficient (r(2)) of >or=0.9968 and 1.2-196.0 ng/mL in urine. The limit of quantification achieved with this method was 0.12 ng/mL in plasma and 1.2 ng/mL in urine. The intra- and inter-assay precision and accuracy values were found to be within the assay variability limits as per the FDA guidelines. The developed assay method was successfully applied to a pharmacokinetic study in human volunteers following intravenous administration of digoxin.     

Direct injection LC-MS/MS method for identification and quantification of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine and 3,4-methylenedioxymethamphetamine in urine drug testing

A method based on direct injection of diluted urine for the identification and quantification of amphetamine, methamphetamine, 3,4-methylenedioxymetamphetamine and 3,4-methylenedioxyamphetamine in human urine by electrospray ionisation liquid chromatography-tandem mass spectrometry was validated for use as a confirmation procedure in urine drug testing. Two deuterium labelled analogues, amphetamine-D5 and 3,4-methylenedioxymetamphetamine-D5, were used as internal standards. Twenty microliter aliquots of urine were mixed with 80 microL internal standard solution in autosampler vials and 10 microL was injected. The chromatographic system consisted of a 2.0 mmx100 mm C18 column and the gradient elution buffers used acetonitrile and 25 mmol/L formic acid. Two product ions produced from the protonated molecules were monitored in the selected reaction monitoring mode. The intra- and inter-assay variability (coefficient of variation) was between 5 and 16% for all analytes at 200 and 6000 ng/mL levels. Ion suppression occurred early after injection but did not affect the identification and quantification of the analytes in authentic urine samples. The method was further validated by comparison with a reference gas chromatographic-mass spectrometric method using 479 authentic urine samples. The two methods agreed almost completely (99.8%) regarding identified analytes when applying a 150 ng/mL reporting limit. Four deviating results were observed for 3,4-methylenedioxymethamphetamine and this was due to uncertainty in quantification around the reporting limit. For the quantitative results the slope of the regression lines were between 0.9769 and 1.0146, with correlation coefficients>0.9339. We conclude that the presented liquid chromatographic-tandem mass spectrometric method is robust and reliable, and suitable for use as a confirmation method in urine drug testing for amphetamines.     

Simultaneous quantification of voriconazole and posaconazole in human plasma by high-performance liquid chromatography with ultra-violet detection

A sensitive and selective high-performance liquid chromatographic (HPLC) method with ultra-violet detection has been developed and validated for the simultaneous determination of posaconazole and voriconazole, two systemic anti-fungal agents. An internal standard diazepam was added to 100 microL of human plasma followed by 3 mL of hexane-methylene chloride (70:30, v/v). The organic layer was evaporated to dryness and the residue was reconstituted with 100 microL of mobile phase before being injected in the chromatographic system. The compounds were separated on a C8 column using sodium potassium phosphate buffer (0.04 M, pH 6.0): acetonitrile:ultrapure water (45:52.5:2.5, v/v/v) as mobile phase. All compounds were detected at a wavelength of 255 nm. The assay was linear and validated over the range 0.2-10.0 mg/L for voriconazole and 0.05-10.0 mg/L for posaconazole. The biases were comprised between -3 and 5% for voriconazole and -2 and 8% for posaconazole. The intra- and inter-day precisions of the method were lower than 8% for the routine quality control (QC). The mean recovery was 98% for voriconazole and 108% for posaconazole. This method provides a useful tool for therapeutic drug monitoring.     

Short-term effects of 3,4-methylen-dioxy-metamphetamine (MDMA) on 5-HT(1A) receptors in the rat hippocampus

The first effects of 3,4-methylen-dioxy-metamphetamine (MDMA, “ecstasy”), on serotonin 1A (5-HT_1A) receptors in rat hippocampus were determined by means of [³H]-8-hydroxy-dipropylamino-tetralin ([³H]-8-OH-DPAT) and 5′guanosine-(γ-[³⁵S]-thio)triphosphate ([³⁵S]-GTPγS) binding as well as inhibition of forskolin (FK)-stimulated adenylyl cyclase (AC) activity. The study was completed by [³⁵S]-GTPγS functional autoradiography experiments carried out in frontal sections of rat brain, including the hippocampal region. Results showed that MDMA was either able to displace [³H]-8-OH-DPAT binding (K_i  500 nM) or to reduce the number of specific sites (B_max) without affecting K_d. The drug also failed to change the [³⁵S]-GTPγS binding or to inhibit AC velocity, underlying its behavior as a non-competitive 5-HT_1A receptor antagonist. Further, MDMA (1 or 100 μM), partially antagonized either [³⁵S]-GTPγS binding stimulation of the agonists 5CT and 8-OH-DPAT or the AC inhibition induced by 5CT and DP-5CT. However, in contrast to binding studies, in AC assays the amphetamine displayed an effect also on EC₅₀, always being less potent than the reference antagonist WAY100,635. In functional autoradiography, MDMA behaved either as a partial 5-HT_1A antagonist in limbic areas or, added alone, as an agonist, increasing the coupling signal presumably through 5-HT release from synapses. Interestingly, the selective 5-HT re-uptake inhibitor (SSRI) fluoxetine had no effect on MDMA [³⁵S]-GTPγS binding activation. This latter finding indicates that the amphetamine can release 5-HT via alternative mechanisms to 5-HT transporter binding, probably via membrane synaptic receptors or vesicular transporters. The release of other transmitters is not excluded. Therefore, our results encourage at extending the study of MDMA biochemical profiles, in the attempt to elucidate those amphetamine-induced pathways with a potential for neurotoxicity or psycho-stimulant activity.