Normal-phase liquid chromatography coupled with electrospray ionization mass spectrometry for chiral separation and quantification of clevudine and its enantiomer in human plasma

A new, simple and enantioselective normal-phase liquid chromatography–mass spectrometry method was presented for the quantification of clevudine and its enantiomer in human plasma. A C18 cartridge was used in this method to extract the enantiomers in 200 μL plasma followed by a chiral separation on a cellulose-based LC column with mobile phase consisted of hexane, methanol and ethanol (62:28:10, V/V/V). The eluate was directed to a mass spectrometry through an electrospray ionization interface. A transition of m/z 261.0 to m/z 126.8 was used for monitoring of clevudine and its enantiomer. This method showed good linearity (R > 0.997), precision (<9.6%) and accuracy (within 95.48–105.9%) within a range of 10–1000 ng/mL for the enantiomers and has been applied to the pharmacokinetics study of clevudine capsules in human plasma.

► A sensitive and reliable LC–MS/MS method for the simultaneous determination of l-FMAU and d-FMAU in human plasma has been developed and validated.
► LLOQ of 10 ng/mL was achieved using only 200 μL of plasma.
► It is the first method for enantioselective determination of FMAU in human plasma.