Simultaneous stereoselective analysis of naftopidil and O-desmethyl naftopidil enantiomers in rat feces using an online column-switching high-performance liquid chromatography method.

A novel online column-switching chiral high-performance liquid chromatography method was developed and validated for the simultaneous determination of naftopidil (NAF) and its O-desmethyl metabolites (DMN) enantiomers in rat feces. Direct and multiple injections of supernatant from rat feces homogenate were allowed through the column-switching system. Analyte extraction was performed on the Capcell Pak mixed-functional column by acetonitrile-phosphate buffer (pH 7.4; 10 mm; 8:92, v/v) flowing at 1 mL/min. Separation of NAF and DMN enantiomers was achieved on the Chiralpak IA column by methanol-acetonitrile-acetate buffer (pH 5.3; 5 mm; 45:33:22, v/v/v) flowing at 0.5 mL/min. The analytes were measured with a fluorescence detector at 290 nm (λ(ex)) and 340 nm (λ(em)). The validated method showed a good linearity [22.5-15,000 ng/mL for (+)-/(-)-NAF; 35-25,000 ng/mL for (+)-/(-)-DMN] and the lowest limits of quantification for NAF and DMN enantiomers were 22.5 and 35 ng/mL, respectively. Both intra- and inter-day variations were <10%. The assay was successfully applied to the fecal excretion of NAF and DMN enantiomers in rat after single oral administration of (±)-NAF. Nonstereoselective excretion of (+)- and (-)-NAF was found in feces, while stereoselective excretion of (+)- and (-)-DMN was observed with higher excretion levels of (+)-DMN, indicating that there may exist stereoselective metabolism for NAF enantiomers.