Identification of Amiodarone Metabolites in Human Bile by Ultraperformance Liquid Chromatography/Quadrupole Time-of-Flight Mass Spectrometry

Abstract

ABSTRACT:Amiodarone is recognized as an effective drug in the treatment of arrhythmias. Previous experiments demonstrated that mono-Ndesethylamiodarone (MDEA) was the major circulating metabolite in humans. In addition, dealkylation, hydroxylation, and deamination were minor metabolic pathways. The purpose of this study was to identify the metabolites of amiodarone in the bile obtained from patients with T-tube drainage after oral drug administration. Amiodarone metabolism in vitro was also investigated using human liver microsomes (HLMs) and S9 fraction. Ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF MS) revealed 33 metabolites in human bile, including 22 phase I and 11 phase II metabolites. The major metabolites were MDEA (M7) and -carboxylate amiodarone (M12). Metabolite M12 was isolated from human bile, and the chemical structure was confirmed using UPLC-Q/TOF MS and 1 H NMR.Moreover, the authentic standards of two hydroxylated metabolites, 2-hydroxylamiodarone and 3-hydroxylamiodarone, were obtained through microbial transformation. Several novel metabolic pathways of amiodarone in human were proposed, including -carboxylation, deiodination, and glucuronidation. The in vitro study demonstrated that incubation of HLMs with amiodarone did not give rise to any carboxyl metabolites. In contrast, M12 and its metabolites were detected in human liver S9 incubation samples, and the production of these metabolites were inhibited almost completely by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, suggesting the involvement of alcohol dehydrogenase in the -carboxylation of amiodarone. Overall, UPLC-Q/TOF MS analysis leads to the discovery of several novel amiodarone metabolites in human bile and underscores the importance of bile as an excretion pathway.

Identifying a Selective Substrate and Inhibitor Pair for the Evaluation of CYP2J2 Activity

“…In this study, NMR analysis revealed that the hydroxylated metabolite is a mixture of two distinct products, 4-and 3-hydroxyamiodarone. These two metabolites have been recently identified in biliary excreta in human subjects after amiodarone dosing (Deng et al, 2011). Deng et al (2011) identified 33 metabolites derived from amiodarone excreted in human bile by mass spectral fragmentation patterns or confirmation by comparison of chromatographic retention times and mass spectra with available reference standards.…”

“…These two metabolites have been recently identified in biliary excreta in human subjects after amiodarone dosing (Deng et al, 2011). Deng et al (2011) identified 33 metabolites derived from amiodarone excreted in human bile by mass spectral fragmentation patterns or confirmation by comparison of chromatographic retention times and mass spectra with available reference standards. Our work confirms the formation of the 4-and 3-hydroxyamiodarone by CYP2J2 in HLM, and it is likely that the former metabolite can be made in extrahepatic tissues that may contribute to the overall in vivo formation.…”

High-Resolution Mass Spectrometry for Characterizing the Metabolism of Synthetic Cannabinoid THJ-018 and Its 5-Fluoro Analog THJ-2201 after Incubation in Human Hepatocytes

“…3C). We propose M9 as 4Ј-OH-THJ-018 because -hydroxylation and -1-hydroxylation were usually the primary biotransformations for drugs with aliphatic side chains and displayed similar physicochemical properties (22)(23)(24). This phenomenon also was observed for 5Ј-OH-JWH-018 and 4Ј-OH-JWH-018 (11 ).…”

Metabolism and Pharmacokinetics of 3-n-Butylphthalide (NBP) in Humans: The Role of Cytochrome P450s and Alcohol Dehydrogenase in Biotransformation

“…Frozen microbial stock culture of C. blakesleana ATCC9244 was first incubated in 250-ml flasks containing potato dextrose agar at 28°C for 7 days in a rotary shaker set to 220 rpm (Asha and Vidyavathi, 2009;Deng et al, 2011). The seed culture (5.0 ml) was then inoculated into 250-ml flasks containing 50 ml of biotransformation medium consisting of dextrose (1.0 g), yeast extract (0.25 g), peptone (0.25 g), NaCl (0.25 g), and K 2 HPO 4 (0.25 g).…”