In vivo metabolism for the hydroxylation of FK778 to the metabolite M3 in humans studied by enantioselective liquid chromatography/tandem mass spectrometry.

A major active metabolite of malononitrilamide FK778 (an immunosuppressant under development) is labeled M3. Due to a chiral center created during in vivo metabolism, the exploration of enantiomer profiles in clinical samples is critical to the characterization of the immunosuppressive activity of M3. An enantioselective liquid chromatography method with detection by tandem mass spectrometry (LC/MS/MS) was developed for the resolution of M3 enantiomers. It was experimentally confirmed that no interconversion between the two enantiomers occurred during sample preparation. This new approach was applied to measure the enantioselectivity of the M3 metabolite in human plasma samples from kidney transplanted patients. The assay results of 91 in vivo human samples from three subjects showed a ratio of 57:43 for the (-)-enantiomer (the 2nd eluter) vs. the (+)-enantiomer (1st eluter), indicating that the enantiometabolism of FK778 through human enzymes is essentially non-specific.