Simultaneous Quantification of a Novel β-Carboline Alkaloid Derivative and Its Three Metabolites in Rat Plasma by UHPLC-MS/MS and Its Application to a Pharmacokinetic Study

1-Methyl-9-(3-pyridinylmethyl)-9H-pyrido[3,4-b] indole (HMYB) is a novel semi-synthetic alkaloid derived from β-carboline harmane, which has been proven to be effective in treatment of encopresis. But the pharmacokinetic characteristics of HMYB were not yet illustrated. In this study, three metabolites (harmane, harmol, and 7-OH-HMYB) of HMYB were isolated from rat urine after oral administration of HMYB and authenticated with nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. Most significantly, HMYB and its three metabolites were simultaneously quantified accurately in rat plasma using a quick, sensitive and selective UHPLC-MS/MS method. On an ACQUITY UPLC BEH C18 column (2.1 × 50 mm, 1.7 µm), the analytes and internal standard (osalmid) were separated by gradient elution using acetonitrile and water containing 0.1% formic acid at a flow rate of 0.4 mL/min. The mass spectrometry detector was used in positive ionization mode for multiple reaction monitoring. The method showed good linearity over the concentration range of 0.5–500 ng/mL for HMYB and three metabolites. The method was applied to the pharmacokinetic study of HMYB and its three metabolites after oral doses of HMYB (25, 50, and 100 mg/kg) and intravenous administration of 0.5 mg/kg in rats. Pharmacokinetic differences between HMYB and its three metabolites were determined, along with the absolute bioavailability of HMYB. These results would be helpful in understanding the efficacy of HMYB. The results will also contribute to providing guidance for the structural modification of more effective semi-synthetic derivatives.