Metabolite Identification of clopamide in rats by UHPLC-Q-Orbitrap MS/MS

Abstract

Clopamide (4-chloro-N-(2,6-dimethyl-1-piperidyl)-3-sulfamoyl-benzamide), a diuretic drug containing a piperidine ring, has been widely used for the treatment of hypertension and edema. No metabolism study has been reported yet. The objective of this study is to investigate clopamide metabolites by using rat liver microsomes (RLM) and administering clopamide (12 mg/kg) to rats. The urine samples were collected until 168 hr and the samples were prepared by liquid-liquid extraction. The phase I metabolites were identified by using both positive and negative ionization modes of a high resolution UHPLC-Q-Orbitrap MS/MS (Q-Exactive). Full scan and dd-MS/MS modes were used to obtain structural information of the metabolites and their structures with plausible metabolic pathways were suggested. We have characterized 10 metabolites of clopamide with their isomers: mono-hydroxylation (M1), dehydrogenation (M2), demethylation (M3), mono-hydroxylation of dehydrogenated metabolite (M4), the addition of mono-aldehyde in the dehydrogenated piperidine ring (M5), the addition of carboxyl group in the dehydrogenated piperidine ring (M6), di-hydroxylation (M7), the addition of carboxyl group (M8), 2,6-demethylation (M9), 2,6-di-hydroxylation in the demethylated piperidine ring (M10). A semi-quantification study was also performed to evaluate long and short-term metabolites from urinary excretion profiling. This metabolism study is carried out for the first time to provide a novel insight into the biotransformation of clopamide that may be useful for drug design and development process as well as doping analysis.