A chip-based @entity11670 in vitro metabolism assay coupled with ESI- @entity15 and ESI- @entity15 / @entity15 detection is described in this paper. The chips were made of a cyclic olefin polymer using a hot embossing process. The introduction of reagent solutions into the chip was carried out using fused- @entity3440 capillaries coupled to two syringes with the flow rate controlled by a syringe pump. Initial experiments described here employed a small commercial guard column in an off-chip format to desalt and concentrate the products of the enzymatic reaction prior to ESI- @entity15 analysis. The system was used both to yield the Michaelis constant (K(m)) of the @entity11670 biotransformation of @entity2645 into @entity5829 and to determine the IC50 value of a chemical inhibitor ( @entity5832 ) for this @entity4433 -mediated reaction. The results demonstrated that the kinetics of the reaction inside the 4-microL volume within the channels of the cyclic olefin polymer chip provided results in agreement with those reported in the literature using conventional assays. The above reactions were carried out using @entity1 liver microsomes, and the metabolites were detected by ESI- @entity15 showing the potential of the chip-based @entity11670 reaction for metabolite screening studies as well as for @entity11670 inhibition assays. A porous monolithic column was subsequently integrated into the chip to perform the reaction mixture cleanup process in an integrated fashion on the chip that is necessary for ESI- @entity15 detection. The miniature monolithic SPE column was prepared in situ inside the chip via UV-initiated polymerization. The results obtained using the integrated system demonstrated the possibility of performing @entity11670 enzymatic reactions in a microvolume reaction chamber coupled directly to ESI- @entity15 detection and required less than 4 microg of @entity35216 protein.

Chip-based XXXX drug metabolism coupled to electrospray ionization-mass spectrometry detection.

@entity1 :: ('9606', 'Species') :: ['human'] @entity11670 :: ('1555', 'Gene') :: ['P450'] @entity35216 :: ('23762', 'Gene') :: ['HLM'] @entity5829 :: ('MESH:D003891', 'Chemical') :: ['desipramine'] @entity4433 :: ('1557', 'Gene') :: ['CYP2C19'] @entity5832 :: ('MESH:D014191', 'Chemical') :: ['tranylcypromine'] @entity2645 :: ('MESH:D007099', 'Chemical') :: ['imipramine'] @entity3440 :: ('MESH:D012822', 'Chemical') :: ['silica'] @entity15 :: ('MESH:D009103', 'Disease') :: ['MS']
@entity11670 :: (1555,Gene) :: ['P450']
