Characterization of in vitro metabolites of three tetrahydroprotoberberine alkaloids in rat liver S9 by high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry.

RATIONALE: Tetrahydroberberine (THB), tetrahydrocoptisine (THCP) and tetrahydrocolumbamine (THCB) belong to the tetrahydroprotoberberine (THPB) alkaloids. Most of them have been extensively studied because of their pharmacological activities such as anti-hypertension, anti-arrhythmia, antimicrobial activity and antioxidant. However, limited information on the pharmacokinetics and metabolism of the three alkaloids has been reported. The purpose of the study was to investigate the in vitro metabolism of THB, THCP and THCB in rat liver S9 by using a rapid and accurate high-performance liquid chromatography-quadrupole time-of-fight mass spectrometry (HPLC/QqTOF-MS) method.

METHODS: The incubation mixture was processed with 15% trichloroacetic acid. Chromatographic separations of three THPB alkaloids and their metabolites was achieved by HPLC/QqTOF-MS and accurate mass measurements of metabolites were automatically performed through data-dependent acquisition in only a 30-min analysis. The detailed structural elucidations of these metabolites were performed by comparing their changes in accurate molecular masses, elemental compositions and product ions with those of the parent drug.

RESULTS: Five, five and four metabolites of THB, THCP and THCB were identified in rat liver S9, respectively. The results show that tO-demethylenation of the 9,10-vicinal methoxyl group was the main metabolic pathway of THB and THCB and that t demethylenation of the two methylenedioxy groups was the main metabolic pathway of THCP. In addition, minor oxidation and methylation reactions could occur for these alkaloids in rat liver S9.

CONCLUSION: This was the first ever investigation of in vitro metabolism of THB, THCP and THCB in rat liver S9 by using a sensitive and accurate HPLC/QqTOF-MS method. The tentatively proposed metabolic pathways of the three alkaloids will provide a basis for further studies of the in vivo metabolism of the three compounds in animals and humans.