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 rat brain.

3,4-Methylenedioxymethamphetamine (MDMA) is a psychoactive drug with abuse liability and neurotoxic potential. Mechanisms by which MDMA produces behavioral and neurotoxic effects have yet to be elucidated. By measuring concentrations of MDMA and its metabolites in relevant brain sites, it may be possible to gain insight into mechanisms underlying MDMA actions. For this purpose, an LC-MS assay with electrospray ionization was developed after homogenization of rat brain and enzymatic conjugate cleavage. The method was successfully validated with respect to selectivity, linearity, accuracy, precision, recovery, and matrix effect and its use should help to delineate the neurotoxic mechanism of action of MDMA.

The demethylenation of methylenedioxymethamphetamine ("ecstasy") by debrisoquine hydroxylase (CYP2D6).

The metabolism of methylenedioxymethamphetamine (MDMA, "ecstasy") was examined in a microsomal preparation of the yeast Saccharomyces cerevisiae expressing human debrisoquine hydroxylase, CYP2D6. Only one product, dihydroxymethylamphetamine (DHMA), was detected in the incubation mixture, and this product accounted for all of the substrate consumption at low concentration (10 microM). Mean +/- SD values of apparent Km(microM) and Vmax (nmol/min per nmol P450) for the demethylenation of (+) and (-)-MDMA at low concentrations (1-100 microM) were 1.72, 0.12 and 6.45, 0.10 and 2.90, 0.10 and 7.61, 0.06, respectively. At high concentrations (> 1000 microM) substrate inhibition was noted, with Ki values of 14.2 and 28.2 mM, respectively, for the (+) and (-) enantiomers. Incubation of MDMA isomers with human liver microsomes indicated that their demethylenation is deficient in the poor metabolizer phenotype. Thus, MDMA is converted to the catecholamine DHMA by CYP2D6, and this may give rise to genetically-determined differences in toxicity.

On the role of alpha-methyldopamine in the antihypertensive effect of alpha-methyldopa.

In renal hypertensive rats the cerebral concentration of alpha-methyldopa, alpha-methyldopamine, alpha-methylnoradrenaline, dopamine and noradrenaline as well as the blood-pressure were determined simultaneously. The antihypertensive effect followed a time course identical to that of the increase in the cerebral concentration of alpha-methyldopamine and of the decrease in the concentration of dopamine, whereas lowering of blood pressure on the one hand, and changes in the levels of alpha-methylnoradrenaline and noradrenaline, on the other, were not related to each other. Dose-response relationships showed the same correlations and lack of correlations, respectively. These results suggest that non-betat-hydroxylated catecholamines play a major role in mediating the antihypertensive effect of alpha-methyldopa or, alternatively, that only the newly biosynthesized alpha-methyl-noradrenaline is effective in lowering blood pressure.

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.

Methylenedioxy designer drugs: mass spectrometric characterization of their glutathione conjugates by means of liquid chromatography-high-resolution mass spectrometry/mass spectrometry and studies on their glutathionyl transferase inhibition potency.

Methylenedioxy designer drugs of abuse such as 3,4-methylenedioxymethamphetamine (MDMA) can be selectively toxic to serotonergic neurons and glutathione (GSH) adducts have been implicated in its neurotoxicity. The catecholic demethylenyl metabolites of MDMA, 3,4-dihydroxymethamphetamine and 3,4-dihydroxyamphetamine, are metabolically oxidized to the corresponding ortho-quinones, which are highly reactive intermediates. These intermediates can then be conjugated with GSH preventing cellular damage. Furthermore, glutathionyl transferase (GST) activity was described to be irreversibly inhibited by the catechols dopamine, α-methyldopa and their GSH conjugates. Therefore, the aims of the present work were the detection and characterization of GSH conjugates of ten methylenedioxy drugs of abuse and their phase I metabolites as well as to assess their inhibition potency on GST activity. The substrates were incubated using human placental GST with or without preincubation by cytochrome P450 enzymes preparations. GST inhibition was tested using chlorodinitrobenzene GSH conjugation as marker reaction. GSH conjugates were analyzed and characterized using LC-high-resolution-MS/MS. For confirmation of postulated fragmentation patterns, formation of GSH conjugates of selected deuterated analogs (deuterated analogue approach, DAA) of the investigated drugs was explored. For the methylenedioxy amphetamines the following steps could be identified: conjugation of the parent compounds at position 2, 5, 6, of the demethylenyl metabolites at position 2 and 5, and of the further deaminated demethylenyl metabolites at position 2. For the ß-keto-phenylalkylamine and pyrrolidinophenone, conjugation of the demethylenyl metabolites and of the deaminated demethylenyl metabolites at position 2 could be identified. The DAA allowed the differentiation of the 2 and 5/6 isomers by confirmation of the postulated mass spectral fragments. Finally, the tested drugs and phase I metabolites showed no inhibition potency on GST activity.

[Identification of 4-O-methyldopamine in rat tissues by reverded-phase liquid chromatography (author's transl)]. / Identification de la 4-O-méthyl dopamine dans les tissus de rat par chromatographie liquide en phase inverse.

4-O-Methyldopamine was identified and assayed in tissues from L-dopa treated rats by reversed-phase high-performance liquid chromatography. The initial steps in the separation of catecholamines were performed by alumine, a weak cation-exchange resin, and thin-layer chromatographic techniques. After L-[3 H] dopa administration, the radiochromatogram was superimposed on the fluorochromatogram obtained with authentic marker 4-O-methyldopamine. This metabolite was detected in kidney but not in brain. The 4-O-methyldopamine:3-O-methyldopamine ratio was 0.032 in kidney. The influence of various treatments on this ratio was investigated. A 160% increase was found after L-dopa administration. This effect was potentiated by nialamide pretreatment (550% increase).

Endogenous synthesis of N-methylnorsalsolinol in rat brain during in vivo microdialysis with epinine.

The in vivo metabolic pathway for the synthesis of N-methylnorsalsolinol, an analogue of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), was studied in the rat brain. N-Methyldopamine (epinine) was perfused at the striatum of the rat brain by in vivo microdialysis. N-Methylnorsalsolinol (NMNSAL) was identified in the brain dialysate after epinine perfusion using gas chromatography-selected-ion monitoring mass spectrometry (GC-SIM-MS). We demonstrated that NMNSAL could be synthesized from epinine with an aldehyde by the Piclet-Spengler condensation reaction in the rat brain.

Analysis of epinine and its metabolites in man after oral administration of its pro-drug ibopamine using high-performance liquid chromatography with electrochemical detection.

Ibopamine (N-methyldopamine O,O'-diisobutyrol ester, hydrochloride) is an ester prodrug of epinine. Epinine is a cardiovascular agent used in congestive heart failure because of its dopaminergic and adrenoreceptor agonist properties. Quantitative analytical methods, using high-performance liquid chromatography coupled with electrochemical detection, were developed for the determination of epinine and its known metabolites in biological media. Epinine was extracted from human plasma and urine via an alumina adsorption procedure; this procedure was also used to estimate epinine conjugates after prior enzymatic hydrolysis. Penicillamine was added to the incubation mixture to inhibit isoquinoline production. Urinary dihydroxyphenylacetic acid levels were obtained using the same alumina adsorption procedure, while a separate analytical procedure utilizing a direct high-performance liquid chromatographic analysis of samples was developed for homovanillic acid and its conjugates. Coefficients of variation for all the assays were below 8%. These methods were used to study the pharmacokinetics and metabolic fate of epinine after oral administration of ibopamine to healthy volunteers.

Determination of d-isoproterenol sulphate by high-performance liquid chromatography with amperometric detection.

The application of reversed-phase high-performance liquid chromatography (HPLC) to the determination of isoproterenol sulphate in human plasma and urine was investigated. Sulphoconjugation of the inactive isomer of isoproterenol was chosen as an experimental model to study individual variations in the rate of sulphation of phenols in man. This approach allowed the ingestion of relatively large amounts of drug and detection of the conjugated material after acid hydrolysis, using alumina clean-up and HPLC with amperometric detection. This method was found to be rapid, sensitive, precise and suited to pharmacokinetic studies in man.

Synthesis and chemical properties of ibopamine and of related esters of N-substituted dopamines--synthesis of ibopamine metabolites.

The therapeutic usefulness of intravenously infused dopamine in congestive heart failure and in shock prompted us to synthesize a wide series of 3,4-O-diesters of dopamine and N-substituted derivatives to obtain an orally active dopamine-like prodrug having adequate absorption and duration of action. The pharmacological results and in particular, the hemodynamic studies in the dog led to the selection of ibopamine, i.e. the 3,4-diisobutyryl ester of N-methyldopamine and to its development as a useful drug for the chronic treatment of congestive heart failure. The choice of ibopamine from among several analogs was also influenced by other favourable properties such as good chemical stability in pharmaceutical formulations and in the biopharmaceutical phases of the absorption, and fast enzymatic activation of the prodrug by plasma and peripheral tissue esterases; the latter property appeared desirable to avoid any accumulation in the central nervous system and consequent undesired side effects. The isomeric mixture of 3-O- and 4-O-isobutyrates of N-methyldopamine as well as the main conjugated metabolites, i.e. the 3-O- and 4-O-sulphate and 4-O-beta-glucuronide of N-methyldopamine were synthesized as analytical references in metabolic studies and for the investigation on their pharmacokinetic and pharmacological properties. Dopamine O-sulphates were also prepared using the methods developed for the corresponding N-methyl derivatives.

Reversed-phase ion-pair partion chromatography of biogenic catecholamines and their alpha-methyl homologues with tributylphosphate as stationary phase.

Ion-pair partition chromatography is applied to the separation of the biogenic catecholamines and their alpha-methyl homologues. A useful selectivity has been obtained using an adduct-forming organic stationary phase (tributylphosphate). The retention of the compounds can be regulated easily by means of the concentration of the counter-ion (the perchlorate ion) in the mobile phase. The selectivity for separation of amines from amino acids can be influenced by changing the pH of the aqueous phase. The phase system shows a good long-term stability and reproducibility with respect to the capacity ratios and the efficiency.

High-performance liquid chromatographic analysis of biogenic amines in cells and in culture media using on-line dialysis and trace enrichment.

A highly sensitive method is presented for the automatic quantitative detection of DOPA metabolites in low concentrations in cells derived from the neural crest using reversed-phase HPLC in combination with fluorescence and electrochemical detection. The HPLC system was combined with on-line dialysis and on-line trace enrichment for the detection of small quantities of DOPA metabolites in culture media. Parameters like detector settings, pH, dialysis time and flow-rates are evaluated and optimized. Static-continuous dialysis can be performed at a low flow-rate concomitant with a high dialysis efficiency (up to >65%) depending on the type of DOPA metabolite. Counterflow dialysis can be used to analyse, with low efficiencies (17-29%), samples consisting of large volumes. Samples containing up to at least 7% (w/v) protein can be analysed in the low flow-rate static-continuous mode. In this last mode of dialysis, limits of detection for dopamine, norepinephrine, epinephrine and n-methyldopamine in DMEM/HAMF12 medium samples are 100 fmol or even lower. Serotonin is detectable at 10 fmol at a signal/noise ratio of 3. Biogenic amines were detectable at a concentration of 10 fmol/microl in a volume of 100 microl medium with an intra- and inter-assay imprecision <6.4%. This method is applied to study the differentiation level of tumour cells in culture and slices of a tumour derived from the neural crest. With this system, we also detected the excretion of DOPA metabolites from PC-12 cells after treatment with prenylamine.

Synthesis and capillary electrophoretic analysis of enantiomerically enriched reference standards of MDMA and its main metabolites.

Enantiomerically-enriched (S)-3,4-methylenedioxymethamphetamine (MDMA) and its main metabolites (S)-4-hydroxy-3-methoxymethamphetamine (HMMA) and (S)-3,4-dihydroxymethamphetamine (HHMA) were prepared for unequivocal identification of the differential enantioselective metabolism of these compounds as well as for its application in the analysis of biological samples. Capillary electrophoresis with cyclodextrin derivatives and a chemical correlation of (S)-MDMA, (S)-HMMA and (S)-HHMA has been performed to assign the absolute stereochemistry of major isomers in analytical standards enriched with such enantiomers.