Investigation of two-dimensional high performance liquid chromatography approaches for reversed phase resolution of warfarin and hydroxywarfarin isomers

•Several offline and online 2D RPLC methods to separate a multicomponent mixture were developed.•A standard mixture of 12 warfarins and monohydroxylated isomers were resolved in 16.2 min.•Online two-dimensional LC method by chiral RP-HPLC × achiral RP-UHPLC provided the fastest separation of all isomers.

Several offline and online 2D HPLC methods were investigated for the reversed phase resolution of a complex mixture of closely related warfarin and hydroxywarfarin isomers. By combining reversed phase achiral/chiral HPLC separation with UV-triggered fraction collection and subsequent chiral/achiral reversed phase HPLC analysis of collected fractions, complete resolution of all 12 components of the mixture was possible. In addition, a faster method was developed from online 2D HPLC analysis where multicomponent fractions from the first dimension are simultaneously chromatographed in the second dimension.