Development of a two-dimensional liquid chromatography system for isolation of drug metabolites.

The separation, isolation and identification of drug metabolites from complex endogenous matrices like urine, plasma and tissue extracts are challenging tasks. Metabolites are usually first identified by mass spectrometry and tentative structures proposed from product ion spectra. In many cases mass spectrometry cannot be used to determine positional isomers and metabolites have to be fractionated in microgram amounts for analysis by NMR. To overcome the difficulties associated with separation and isolation of drug metabolites from biological matrices, a new two-dimensional liquid chromatography system has been developed. The retention times of 45 acidic, basic and neutral compounds were determined on liquid chromatographic columns with different stationary phases in order to identify two columns with highly different selectivity to be used for two-dimensional liquid chromatography. Drug metabolites of three model compounds were first generated in vitro with liver microsomes and then compared with potential metabolites formed by oxidation with hydrogen peroxide catalyzed by meso-tetra (4-sulphonatophenyl) porphine (porphine). The results showed that the porphine system could be used as a complementary system for the generation of phase I microsomal metabolites with high yield of some metabolites in a less complex matrix. The two-dimensional liquid chromatography system was used to separate and isolate microsomal and porphine generated drug metabolites in off-line and on-line mode. Finally, to verify the utility of the developed system, urine samples were spiked with metabolite standards of model compounds for separation in the two-dimensional system. Excellent separations were obtained with an amide column in the first dimension and a pentafluorophenylpropyl (PFPP) column in the second dimension. The metabolites were successfully separated from each other as well as from the complex biological matrix. The results demonstrate the applicability of the system for fractionation of drug metabolites but it could also be used in many other analytical purposes, especially for basic compounds. Trace levels of metabolites were successfully separated in the on-line mode which failed in the off-line mode.