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.
Stereochemical analysis of 3,4-methylenedioxymethamphetamine and its main metabolites in human samples including the catechol-type metabolite (3,4-dihydroxymethamphetamine).
3,4-Methylenedioxymethamphetamine (MDMA; "ecstasy") is a designer drug commonly misused in large segments of young populations. MDMA is usually formulated in tablets of its racemate (1:1 mixture of its enantiomers) in doses ranging from 50 to 200 mg. MDMA has an enantioselective metabolism, the (S)-enantiomer being metabolized faster than the (R)-enantiomer. Different pharmacologic properties have been attributed to each enantiomer. The carbon responsible for MDMA chirality is preserved along its metabolic disposition. An analytical method has been developed to determine MDMA enantiomers and those from its major metabolites, 3,4-methylenedioxyamphetamine (MDA), 3,4-dihydroxymeth-amphetamine (HHMA), and 4-hydroxy-3-methoxymethamphet-amine (HMMA). It has been applied to the analysis of plasma and urine samples from healthy recreational users of MDMA who participated voluntarily in a clinical trial and received 100 mg (R,S)-MDMA. HCl orally. (R)/(S) ratios both in plasma (0-48 h) and urine (0-72 h) for MDMA and MDA were >1 and <1, respectively. Ratios corresponding to HHMA and HMMA, close to unity, deviate from theoretical expectations and are most likely explained by the ability of MDMA to autoinhibit its own metabolism. The short elimination half-life of (S)-MDMA (4.8 h) is consistent with the subjective effects and psychomotor performance reported in subjects exposed to MDMA, whereas the much longer half-life of the (R)-enantiomer (14.8 h) correlates with mood and cognitive effects experienced on the next days after MDMA use.
Binding characteristics of brivaracetam, a selective, high affinity SV2A ligand in rat, mouse and human brain: relationship to anti-convulsant properties.
Brivaracetam is a novel synaptic vesicle protein 2A (SV2A) ligand reported to be 10 fold more potent than levetiracetam in animal models of epilepsy. This study reports the binding profile of brivaracetam in the brain of several species in relation to its anticonvulsant properties. The affinity, kinetics and selectivity of brivaracetam and its tritiated form [(3)H]ucb 34714 have been determined by in vitro binding experiments in rat, human and mouse brain and on recombinant human SV2A. Brivaracetam and levetiracetam ex vivo binding to SV2A and anticonvulsant activities in audiogenic mice were compared in relation to dose and time. Brivaracetam bound selectively with 20 fold higher affinity than levetiracetam to SV2A. [(3)H]ucb 34714 bound reversibly and with high affinity to an homogenous population of binding sites in rat and human brain and to human SV2A expressed in CHO cells. The binding sites labeled by [(3)H]ucb 34714 in brain had the pharmacological characteristics of SV2A and no specific binding could be detected in the brain of SV2A(-/-) knock-out mice. The time- and dose-dependency of brivaracetam and levetiracetam for binding to brain SV2A and for providing seizure protection in audiogenic mice correlated well; brivaracetam being more potent and faster than levetiracetam. Brivaracetam is a potent and selective SV2A ligand. From its affinity and pharmacokinetics, simulations predicted that at therapeutically relevant doses, brivaracetam should occupy more than 80% of SV2A in human brain, in line with levels of occupancy observed in pre-clinical models of epilepsy.