Chip-Based P450 Drug Metabolism Coupled to Electrospray Ionization-Mass Spectrometry Detection

Abstract

A chip-based P450 in vitro metabolism assay coupled with ESI-MS and ESI-MS/MS 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-silica 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-MS analysis. The system was used both to yield the Michaelis constant (K m ) of the P450 biotransformation of imipramine into desipramine and to determine the IC50 value of a chemical inhibitor (tranylcypromine) for this CYP2C19-mediated reaction. The results demonstrated that the kinetics of the reaction inside the 4-µL 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 human liver microsomes, and the metabolites were detected by ESI-MS showing the potential of the chip-based P450 reaction for metabolite screening studies as well as for P450 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-MS 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 P450 enzymatic reactions in a microvolume reaction chamber coupled directly to ESI-MS detection and required less than 4 µg of HLM protein.

Small Molecule Quantification by Liquid Chromatography-Mass Spectrometry for Metabolites of Drugs and Drug Candidates

“…These observations indicate that the signal of a metabolite can be higher or lower than that of the substrate and that none of the different metabolic transformations give a consistently better agreement in the slopes. This result does not appear to be the case for carboxylic acid metabolites (Hop et al, 2005;Valaskovic et al, 2006;Ramanathan et al, 2007) or for the loss of an amine by N-dealkylation (Benetton et al, 2003), both of which consistently show a lower signal for the metabolite. Indeed, the fact that all the compounds tested have multiple nitrogen atoms and no other ioniz- b Slope (average) of the standard curve for metabolite versus internal standard divided by slope (average) of the standard curve for substrate versus internal standard using nanospray ionization.…”

“…It has been proposed that low-flow LC-MS techniques such as nanospray may provide less variability in ionization (Benetton et al, 2003;Hop et al, 2005;Ramanathan et al, 2011) (see below) than regular LC-MS. To test this hypothesis we also compared standard curves generated using CSI LC-MS. The results are shown in Table 1, and the overall variability is less than that for normal-flow LC-MS. To look at the absolute deviations associated with the difference in signals from parent and metabolite, we calculated the average of the absolute differences of the ratio of the metabolite and parent signals from 1 (unsigned average difference).…”

“…In this regard, other methodologies show promise. For example, a number of investigators have used nanospray LC-MS to decrease the differences between the signal from parent drug and metabolites (Benetton et al, 2003;Hop et al, 2005;Valaskovic et al, 2006;Ramanathan et al, 2007). Furthermore, a number of other methods to estimate exposure to a metabolite using corrected responses in LC-MS/MS (Vishwanathan et al, 2009;Gao et al, 2010;Ma et al, 2010;Tong et al, 2010;Mutlib et al, 2011;Walker et al, 2011) or NMR based methods (Vishwanathan et al, 2009;Mutlib et al, 2011;Walker et al, 2011) have been developed.…”

Microchip technology in mass spectrometry

“…The coupling of these external emitter needles was typically done by drilling and gluing. In addition to spraying from a sharp and accurately defined nozzle, off-chip ionization enabled easier application of high voltage at the end of the separation channel since, as in conventional ESI/MS interfacing methods, the chips could be coupled to external liquid junctions or sheath liquid interfaces through transfer capillaries (Figeys et al, 1998;Chan et al, 1999;Li et al, 1999Li et al, , 2000bLi et al, , 2008Zhang et al, 1999;Vrouwe et al, 2000;Meng et al, 2001;Kameoka et al, 2002;Benetton et al, 2003;Mao, Chu, & Lin, 2006;Zheng et al, 2007). Separately assembled external microsprayers attached to the microchannel outlet (Deng, Zhang, & Henion, 2001), as well as on-chip liquid junctions and sheath flow channels (Razunguzwa, Lenke, & Timperman, 2005) coupled to external emitters, have also been reported.…”

Microfluidics for multiplexed MS analysis

“…The practical use of the infusion system with the electrospray array chip [30] has been documented on a plethora of applications including drug metabolism studies [27], direct analysis of drugs in bodily fluids [28][29][30] , carbohy- drates and glycoproteins [31][32][33][34] or protein and peptide samples [35,36]. Although direct integration of other functionalities into the chip platform, e.g.…”