Abstract
A simple and direct analysis using column‐switching HPLC method was developed and validated for the quantification of active metabolites of sibutramine, N‐mono‐desmethyl metabolite (metabolite 1, M1) and N‐di‐desmethyl metabolite (metabolite 2, M2) in the serum of rats administered sibutramine HCl (5.0 mg/kg, p.o.). Rat serum was directly injected onto the precolumn without sample prepreparation step following dilution with mobile phase A, i. e., methanol–ACN–20 mM ammonium phosphate buffer (pH 6.0 with phosphoric acid) (8.3:4.5:87.2 by volume). After the endogenous serum components were eluted to waste, the system was switched and the analytes were eluted to the trap column. Active metabolites M1 and M2 were then back‐flushed to the analytical column for separation with mobile phase B, i. e., methanol–ACN–20 mM ammonium phosphate buffer (pH 6.0 with phosphoric acid) (35.8:19.2:45 by volume) and detected at 223 nm. The calibration curves of active metabolites M1 and M2 were linear in the range of 0.1–1.0 μg/mL and 0.15–1.8 μg/mL. This method was fully validated and shown to be specific, accurate (10.4–10.7% error), and precise (1.97–8.79% CV). This simple and rapid analytical method using column‐switching appears to be useful for the pharmacokinetic study of active metabolites (M1 and M2) of sibutramine.