2008
On-line preconcentration of 4-hydroxydiclofenac for MEKC separation by means of sweeping in homogenous electric field
ŘEMÍNEK, Roman; Jiří KONEČNÝ and Zdeněk GLATZBasic information
Original name
On-line preconcentration of 4-hydroxydiclofenac for MEKC separation by means of sweeping in homogenous electric field
Name in Czech
On-line zakoncentrování 4-hydroxydiclofenacu pro MEKC pomocí sweepingu v homogenním elektrickém poli
Authors
ŘEMÍNEK, Roman; Jiří KONEČNÝ and Zdeněk GLATZ
Edition
Brno, Česká republika, Book of Abstracts CECE 2008, p. 50-51, 2 pp. 2008
Publisher
dr. František Foret
Other information
Language
English
Type of outcome
Proceedings paper
Field of Study
10600 1.6 Biological sciences
Country of publisher
Czech Republic
Confidentiality degree
is not subject to a state or trade secret
Organization unit
Faculty of Science
Keywords in English
phase I metabolites; microsomes; drug metabolism; CE methods
Tags
International impact, Reviewed
Changed: 24/9/2009 15:12, prof. RNDr. Zdeněk Glatz, CSc.
In the original language
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.
In Czech
Byla vypracov8na metoda pro on-line zakoncentrování 4-hydroxydiclofenacu pro MEKC pomocí sweepingu v homogenním elektrickém poli. Metoda byla použita pro kineticke studium CYP2C9 při koncentracích nižších než je 2 uM.
Links
| GA203/06/0047, research and development project |
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| LC06023, research and development project |
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| MSM0021622413, plan (intention) |
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