Determination of the active metabolites of sibutramine in rat serum using column-switching HPLC.
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 microg/mL and 0.15-1.8 microg/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.
Um
So Young
SY
Department of Pharmacology, National Institute of Toxicological Research, Korea Food and Drug Administration, Seoul, Korea.
Kim
Kyu-Bong
KB
Kim
Seon Hwa
SH
Ju
Young Cheol
YC
Lee
Hye Sang
HS
Oh
Hye young
Hy
Choi
Ki Hwan
KH
Chung
Myeon Woo
MW
D000284:Administration, Oral; D000818:Animals; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods; D003503:Cyclobutanes / Q000008:administration & dosage / Q000097:blood*; D008297:Male; D015394:Molecular Structure; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013237:Stereoisomerism
D003503:Cyclobutanes; C058254:sibutramine