In vivo metabolism study of diuretics clopamide by LC-MS/MS

Abstract

In vivo metabolism study of diuretics clopamide by LC-MS/MS Farzana Binte Rafique1,2 Anca Raluca Muresan1,2, Khandoker Asiqur Rahaman1,2, and Oh-Seung Kwon1,2* 1Doping Control Center, Korea Institute of Science and Technology, Seoul, 02792, Korea, 2Division of Bio-Medical Science & Technology, KIST School, Korea University of Science and Technology, Seoul 02792, Korea. * Corresponding author: oskwon@kist.re.kr Diuretics are often illegally used as a masking agent by athletes in various kind of sports area, and clopamide is included in the list of prohibited substances of World Anti-Doping Agency. The objective of this study was to investigate clopamide metabolites in rat (Sprague-Dawley) urine both negative ionization ([M-H]-, m/z 344) and positive ionization ([M+H] +, m/z 346) since its metabolism study was not reported yet. In this study, clopamide (12 mg/kg) was administered in rats and collected urine by 168 hour. The metabolites from urine sample were identified by using both negative and positive ionization modes of an ultra high-performance liquid chromatography/Orbitrap mass spectrometer (Q-Exactive). The full scan and dd-MS/MS modes were used to obtain structural information of the metabolites and their possible structures were suggested. We have characterized 8 metabolites and their isomers M1a-d ([M+H+16] +), M2 ([M+H-2] +), M3 ([M+H-14]+), M4a-d ([M+H+14] +), M5 ([M+H+12]+), M6 ([M+H+24]+), M7 ([M+H+32] +), M8 ([M+H+34] +) in positive ionization mode, and 9 metabolites with their isomers M1a-c ([M-H+16]-), M2 ([M-H-2]-), M3 ([M-H-14]-), M4 ([M-H+14]-), M5 ([M-H+12]-), M6 ([M-H+24]-), M7 ([M-H+32]-), M8 ([M-H+34]-), M9 ([M-H+30]-) in negative ionization mode at collision energy of 40 eV. The major biotransformation processes for the phase-1 metabolism were mono-hydroxylation (M1), dehydrogenation (M2), demethylation (M3), mono-hydroxylation and dehydrogenation (M4), addition of mono-aldehyde wi