Development of immobilized enzyme reactors based on human recombinant cytochrome P450 enzymes for phase I drug metabolism studies

Two cytochrome P450 (CYP)-based immobilized enzyme reactors (IMERs) were developed to perform automated on-line phase I drug metabolism studies. For this purpose, biotinylated recombinant CYP2D6 or CYP3A4 reconstituted systems were anchored to the surface of two monolithic mini-columns (2 mm × 6 mm I.D.), which had been covalently grafted with NeutrAvidin. After optimization of immobilization conditions, the obtained IMERs were integrated on-line into a LC hyphenated to an electrospray ionization MS/MS system. Studies with probe substrates and a known competitive inhibitor were performed, showing the potential of CYP-based IMERs in drug metabolism. In the optimized conditions, ca. 15 experiments were carried out with each bioreactor.