Generic On-line Method for Identification of Cytochrome P450 Metabolism Products Employing Capillary Electrophoresis Coupled to Mass Spectrometry
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| Year of publication | 2012 |
| Type | Conference abstract |
| MU Faculty or unit | |
| Citation | |
| Description | Rational drug discovery requires an early appraisal of all factors impacting on the likely success of a drug candidate in the subsequent clinical testing. Besides others, study of metabolism of leads and their affinity to clinically important drug-metabolizing enzymes such as cytochromes P450 (CYP) represents a key step carried out during early stages of the development of new drugs. Capillary electrophoresis (CE) is a promising technique in this field due to its rapid and highly effective separations, minute sample consumption, high throughput by automation and easy electrospray ionization (ESI) interfacing with mass spectrometric detection (MS). The goal of this study was to introduce a method allowing on-line identification of CYP metabolism products. The system combining on-line CE and Q-TOF MS was employed. Whereas on-line CE integrates injection of reactants, incubation of the enzymatic reaction and separation of reaction mixture into a single fully automated run, tandem MS detection enables direct identification of reaction products. Methodology of transverse diffusion of laminar flow profiles developed by Krylov et al. [1] was utilized in order to ensure the generic nature of reactants’ mixing inside the capillary nanoliter-scale reactor. The applicability of presented method was demonstrated using model system of CYP2C9 isoform and its substrates. All the experiments were accomplished on the Agilent 7100 CE and Bruker maXis impact ESI-Qq-TOF MS system. Solutions of CYP and mixture of model substrate and NADPH both prepared in incubation buffer were separately injected into capillary as a series of consecutive plugs by hydrodynamic pressure. Resulting reaction mixture formed by diffusion was incubated in capillary at least for 10 minutes. Electrophoretic separations were carried out by application of 30 kV (positive polarity) in uncoated fused silica capillary (75 cm; 75um) filled with ammonium acetate used as background electrolyte and thermostated at 37 C. CE-ESI-MS coupling was carried out with sheath liquid interface. Sheath liquid solution was composed of methanol/water mixture (50:50) with ionization additive. Identification of substrates and products was enabled due to tandem MS in MRM mode. Principle of reactant’s mixing inside capillary based on diffusion and employment of tandem MS detection guarantee generic applicability of the method regardless of tested CYP isoform and its substrate. |
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