Plasma Direct Injection High-Performance Liquid Chromatographic Method for Simultaneously Determining E3810 Enantiomers and Their Metabolites by Using Flavoprotein-Conjugated Column

A high-performance liquid chromatographic method using the column-switching technique was developed for the simultaneous determination of E3810 and its metabolites (M1-M4). An avidin column was used for in-line pretreatment to exclude plasma proteins, allowing direct injection of a large volume of plasma. A flavoprotein-conjugated chiral stationary phase in a gradient elution mode then gave baseline separation of E3810 and the four metabolites. The enantiomers of E3810 and M3 were also separated. The method is simple, rapid, accurate, and precise. Since no extraction procedure is employed, which might involve different recoveries of different metabolites, no internal standard is necessary. The method was applied to analyze E3810 and its metabolites in plasma after intravenous injection of the drug in beagle dogs.