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The use of capillary electrophoresis for studying the in vitro glucuronidation of 7-hydroxycoumarin

Bogan, Declan P. and Killard, Anthony J. and O'Kennedy, Richard (1995) The use of capillary electrophoresis for studying the in vitro glucuronidation of 7-hydroxycoumarin. Journal of Capillary Electrophoresis, 2 (5). pp. 241-245.

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Abstract

An assay utilizing CE, has been developed for studying the in vitro metabolism of 7-hydroxycoumarin to 7-hydroxycoumarin-glucuronide. A reaction mixture containing a crude preparation of bovine uridine diphosphate (UDP) glucuronyl transferase (UDPGT) and the substrates uridine diphosphate glucuronic acid (UDPGA) and 7-hydroxycoumarin was prepared. Aliquots were removed from the reaction mixture at specific time intervals and analyzed on a 57-cm untreated silica capillary for the presence of 7-hydroxycoumarin-glucuronide. Samples were electrophoresed in a 100 mM phosphate/11 mM deoxycholic acid (sodium salt)/acetonitrile electrolyte buffer, pH 7.0, at 30 kV, with detection at 320 nm. The method allowed the determination of 7-hydroxycoumarin-glucuronide without sample cleanup, with a limit of detection of 2 micrograms/mL, and a linear detection range of 0 microgram/mL to 100 micrograms/mL. The 7-hydroxycoumarin-glucuronide, concentrations were calculated from calibration curves of standards prepared in enzyme solution. The initial rate of production of 7-hydroxycoumarin-glucuronide, in the first 70 min, was 3.1 +/- 0.13 nmol/mL/min/mg protein. The method was fast and reliable with percentage relative standard deviations for concentration of 7-hydroxycoumarin-glucuronide, over time, of less than 10%.

Item Type:Article (Published)
Refereed:Yes
Subjects:Physical Sciences > Chemistry
DCU Faculties and Centres:DCU Faculties and Schools > Faculty of Science and Health > School of Biotechnology
Research Initiatives and Centres > National Centre for Sensor Research (NCSR)
Publisher:ISC Technical Publications, Inc.
ID Code:21
Deposited On:08 Nov 2006 by DORAS Administrator. Last Modified 30 Jan 2009 11:09

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