On-line preconcentration of 4-hydroxydiclofenac for MEKC separation by means of sweeping in homogenous electric field
| Authors | |
|---|---|
| Year of publication | 2008 |
| Type | Article in Proceedings |
| Conference | Book of Abstracts CECE 2008 |
| MU Faculty or unit | |
| Citation | |
| Field | Biochemistry |
| Keywords | phase I metabolites; microsomes; drug metabolism; CE methods |
| Description | Cytochromes P450 (CYP) plays a key role in drug metabolism. Many analytical approaches to follow the drug metabolism by CYP were established. Micellar electrokinetic capillary chromatography (MEKC) is one of the most favorable method, particularly due to a low sample requirement and high efficiency, moreover CYP reaction mixture can be analyzed without any pre-treatment. However, small sample volumes and short optical path-lengths lead to low concentration sensitivity of MEKC. In response to this problem, several on-line focusing methods have been developed to preconcentrate analytes inside the capillary before separation and detection. In this study a method using sweeping in homogenous electric field for on-line preconcentration of 4-hydroxydiclofenac was developed. Subsequently it was used for determination of CYP2C9 kinetic parameters. Main emphasis was put on substrate concentrations bellow 2 uM where a weak positive cooperation was estimated. The 50 um fused silica capillary (56 cm effective length) was used to carry out all separations. 60 mM SDS in 20 mM phosphate 20 mM tetraborate buffer was used as a background electrolyte. Injection was accomplished by an application of 50 mbar pressure to the sample vial for 48 s. Separations were performed at 24 kV (positive polarity) and the temperature of capillary 25 oC. Analytes were detected using diode array detector at 200 nm with a bandwidth 10 nm. Reaction mixtures containing 0,25 25 mM of diclofenac, 11,73 pM recombinant CYP2C9, 1 mM NADP+, 6 mM glucose 6 phosphate, 35 U.ml-1 glucose-6-phosphate dehydrogenase, 5,1 mM MgCl2, 0,1 M KCl and 50 mM phosphate buffer incubated for 30 minutes. Kinetic parameters were evaluated by SigmaPlot 8.02 software. As the result Michaelis constant Km = 4,62 uM, maximum reaction velocity Vmax = 18,35nmol.min-1.nmol-1 and Hill coefficient n = 1,22 (p = 0,05; R2 = 0,991) values were determined. Value of Hill coefficient confirms a presence of weak positive cooperativity in low substrate concentration region. |
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