On-line Capillary Electrophoresis Coupled to Mass Spectrometry: from Enzymatic Reaction to Identification of Reaction Products within Single Analysis
| Authors | |
|---|---|
| Year of publication | 2012 |
| Type | Article in Proceedings |
| Conference | Advances in Chromatography and Electrophoresis & Chiranal 2012 |
| MU Faculty or unit | |
| Citation | |
| Field | Biochemistry |
| Keywords | On-line capillary electrophoresis; mass spectrometry; enzyme reaction |
| Description | Cytochrome P450 enzymes (CYP) play a key role in metabolism of 75 % commonly prescribed drugs in the human body [1]. For this reason, studies of the affinity of a candidate compound to this group of enzymes represent an important part of a new drug development process [2]. Since large compound libraries undergo the testing, there is ongoing trend towards the miniaturization and throughput enhancement of screening assays in order to economize and expedite the whole development process. Capillary electrophoresis (CE) is a promising technique in this field due to its rapid highly effective separations, minute sample consumption, high throughput by automation and easy electrospray ionization (ESI) interfacing with mass spectrometric detection (MS). Moreover, in on-line CE a fused silica capillary is used not only as a separation column but also as a reaction chamber, creating a nanoliter-scale reactor. The goal of this study was to introduce a new generic on-line CE-ESI-MS method allowing identification of drug metabolism products. As was mentioned above, extensive numbers of candidates are tested within early stages of a new drug development process. Therefore, the generic procedure for reactants mixing inside the narrow capillary presented by Krylov et al. was employed [3]. This approach is based on transverse diffusion of reactants injected as parabolic plugs by relatively high hydrodynamic pressure. The reaction of the clinically important cytochrome P450 2C9 isoform (CYP2C9) with diclofenac was chosen as a model system in this study. All the experiments were conducted on the Agilent 7100 CE System and Agilent 6320 Ion Trap MS system enabling tandem MS in time analyses. An uncoated fused silica capillary (75 cm; 75 um) with edged conical tip was thermostated at 37 C. Separations were accomplished by application of 30 kV, (positive polarity, 50 uA current limit) and pressure of 50 mbar. 30 mM ammonium acetate (pH 9.20) was used as a BGE. All reactants were prepared in incubation buffer consisting of 50 mM TRIS-HCl buffer (pH 7.50). Injection procedure was as follows: 2 s of diclofenac and NADPH solution, 2 s CYP2C9 solution, 2 s diclofenac and NADPH solution by pressure of 50 mbar, and 2 s BGE by pressure of 15 mbar. Enzymatic reactions were incubated for 10 min. Method optimization and validation was performed with reactant’s solutions containing 100 uM diclofenac and 4 mM NADPH; 333 nM CYP2C9. CE-ESI-MS coupling was carried out with sheath liquid interface. Sheath liquid consisted of 0.3 % formic acid in methanol/water solution (50/50). Nebulizing gas pressure was 345 mbar. Flow rates of sheath liquid and drying gas were set on 4 uL/min and 4 L/min, respectively. As the result, the on-line CE-ESI-MS method for identification of reaction products provided by CYP2C9 drug biotransformation activity was introduced. Incubation of the enzymatic reaction, separation of reaction products and their identification by tandem MS was integrated into single analysis. Model substrate diclofenac and its metabolite 4’-hydroxydiclofenac were detected in m/z range of 230 – 325 with target masses 296 and 312, respectively. Mass spectrum of 4’-hydroxydiclofenac showing specific pattern with fragments 294 and 266 is depicted in Figure 1. Since diffusion is a characteristic property of all molecules, the presented system can be employed for testing of any putative substrate of CYP2C9. The new method enabling formation and identification of enzymatic reaction products within single analysis was introduced. Principle of reactant’s mixing inside capillary based on diffusion and employment of tandem MS detection guarantee its generic applicability. |
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