Identification of in vitro and in vivo potential metabolites of novel cardiovascular and adrenolytic drugs by liquid chromatography-mass spectrometry with the aid of experimental design

Abstract

Drug metabolism in liver microsomes was studied in vitro using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Relevant drug was incubated with dog, human and rat liver microsomes (DLMs, HLMs, RLMs) along with NADPH, and the reaction mixture was analyzed by LC-MS/MS to obtain specific metabolic profile. GRACE analytical C18 column, Vision HT (50 × 2 mm, 1.5 μm) was implemented with acetonitrile and water (+ 5 mM ammonium acetate) in a gradient mode as the mobile phase at a flow 0.4 mL.min⁻¹. Different phase I and phase II metabolites were detected and structurally described. The metabolism of the studied drugs occurred via oxidation, hydroxylation and oxidative deamination processes. Conjugates with the glucuronic acid and sulfate were also observed as phase II biotransformation. The central composite design (CCD) showed that factors, such as time incubation, liver microsomal enzymes concentration and NADPH concentration, along with drying gas temperature, nebulizer gas pressure and capillary voltage significantly affected the final response of the method. This study describes the novel information about the chemical structure of the potential metabolites of selected biologically active compounds, which provide vital data for further pharmacokinetic and in vivo metabolism studies.