Liquid chromatography-quadrupole time-of-flight mass spectrometry for screening in vitro drug metabolites in humans: Investigation on seven phenethylamine-based designer drugs

Phenethylamine-based designer drugs are prevalent within the new psychoactive substance market. Characterisation of their metabolites is important in order to identify suitable biomarkers which can be used for better monitoring their consumption. Careful design of in vitro metabolism experiments using subcellular liver fractions will assist in obtaining reliable outcomes for such purposes. The objective of this study was to stepwise investigate the in vitro human metabolism of seven phenethylamine-based designer drugs using individual families of enzymes. This included para-methoxyamphetamine, para-methoxymethamphetamine, 4-methylthioamphetamine, N-methyl-benzodioxolylbutanamine, benzodioxolylbutanamine, 5-(2-aminopropyl) benzofuran and 6-(2-aminopropyl) benzofuran. Identification and structural elucidation of the metabolites was performed using liquid chromatography-quadrupole-time-of-flight mass spectrometry. The targeted drugs were mainly metabolised by cytochrome P450 enzymes via O-dealkylation as the major pathway, followed by N-dealkylation, oxidation of unsubstituted C atoms and deamination (to a small extent). These drugs were largely free from Phase II metabolism. Only a limited number of metabolites were found which was consistent with the existing literature for other phenethylamine-based drugs. Also, the metabolism of most of the targeted drugs progressed at slow rate. The reproducibility of the identified metabolites was assessed through examining formation patterns using different incubation times, substrate and enzyme concentrations. Completion of the work has led to a set of metabolites which are representative for specific detection of these drugs in intoxicated individuals and also for meaningful evaluation of their use in communities by wastewater-based drug epidemiology

In vitro hepatic metabolism of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and 2,2',4,4',5-pentabromodiphenyl ether (BDE-99)

Polybrominated diphenyl ethers (PBDEs) are flame retardants that were added to many consumer products and have emerged as persistent and bioaccumulative environmental contaminants. Penta-BDE was a commercial PBDE mixture that was used extensively in North America. 2,2',4,4'-Tetrabromodiphenyl ether (BDE-47) and 2,2',4,4',5-pentabromodiphenyl ether (BDE-99) are the dominant congeners in the Penta-BDE mixture and occur at similar levels in the mixture and in air, dust, and sediments. In contrast, the concentration of BDE-99 is 10-fold lower than that of BDE-47 in most wildlife and human samples, which could be due to more extensive metabolism of BDE-99 than BDE-47 by hepatic cytochrome P450 (CYP) enzymes. To investigate this hypothesis, a liquid chromatography-mass spectrometry based assay was developed and validated to characterize the biotransformation of BDE-99 by liver microsomes. Rat liver microsomes were obtained from animals treated with dexamethasone, phenobarbital, 3-methylcholanthrene or corn oil. Up to six hydroxylated metabolites of BDE-99 were formed by different rat liver microsomal preparations. The major metabolite, 4-hydroxy-2,2',3,4',5-pentabromodiphenyl ether (4-OH-BDE-90), was formed at 2.7 pmol/min/mg protein by liver microsomes obtained from corn oil treated rats. CYP3A1, CYP1A1, and CYP2A2 were the most active rat recombinant CYP enzymes. Incubating BDE-99 with human liver microsomes resulted in the formation of 10 hydroxylated metabolites. The major metabolites were 2,4,5-tribromophenol (2,4,5-TBP), 5ʹ-hydroxy-2,2ʹ,4,4ʹ,5-pentabromodiphenyl ether (5'-OH-BDE-99) and 4'-hydroxy-2,2',4,5,5'-pentabromodiphenyl ether (4'-OH-BDE-101) and their rates of formation ranged between 25 and 45 pmol/min/mg protein. Incubating BDE-47 with human liver microsomes resulted in the formation of 9 hydroxylated metabolites. The major metabolites were 5-hydroxy-2,2',4,4'-tetrabromodiphenyl ether (5-OH-BDE-47) and 6-hydroxy-2,2',4,4'-tetrabromodiphenyl ether (6-OH-BDE-47) and their rates of formation were 23 and 27 pmol/min/mg protein, respectively. CYP2B6 was the major human CYP enzyme responsible for the formation of all the hydroxylated metabolites of BDE-47 and BDE-99. In conclusion, BDE-99 underwent more extensive oxidative metabolism by human than rat liver microsomes and was biotransformed into a different set of hydroxylated metabolites by human than rat liver microsomes. Metabolism of BDE-47 and BDE-99 by human liver microsomes proceeds at similar pace, which suggests that oxidative metabolism does not explain the difference in BDE-47 and BDE-99 blood concentrations in humans.Pharmaceutical Sciences, Faculty ofGraduat