title: 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.
abstract: 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 β-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.
authors.affiliation: Department of Experimental and Clinical Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, Saarland University, Homburg, Saarland, Germany. Electronic address: markus.meyer@uks.eu.
authors.forename: Markus R
authors.initials: MR
authors.lastname: Meyer
authors.affiliation: Department of Experimental and Clinical Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, Saarland University, Homburg, Saarland, Germany.
authors.forename: Lilian H J
authors.initials: LH
authors.lastname: Richter
authors.affiliation: Department of Experimental and Clinical Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, Saarland University, Homburg, Saarland, Germany.
authors.forename: Hans H
authors.initials: HH
authors.lastname: Maurer
mesh_terms: D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D003846:Deoxyepinephrine / Q000031:analogs & derivatives / Q000032:analysis / Q000737:chemistry; D004137:Dinitrochlorobenzene / Q000737:chemistry; D057075:Enzyme Assays; D005260:Female; D005978:Glutathione / Q000737:chemistry*; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D007536:Isomerism; D018817:N-Methyl-3,4-methylenedioxyamphetamine / Q000737:chemistry / Q000378:metabolism*; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D013056:Spectrophotometry, Ultraviolet; D053719:Tandem Mass Spectrometry
chemical_list: D004137:Dinitrochlorobenzene; C069512:alpha-methylepinine; C023492:alpha-methyldopamine; D003577:Cytochrome P-450 Enzyme System; D005982:Glutathione Transferase; D005978:Glutathione; D018817:N-Methyl-3,4-methylenedioxyamphetamine; D003846:Deoxyepinephrine
keywords: 3,4-Methylenedioxymethamphetamine; Glutathione; Glutathione conjugates; High resolution mass spectrometry; Methylenedioxy designer drugs