In vitro glucuronidation of 7-hydroxycoumarin and determination of 7-hydroxycoumarin and 7-hydroxycoumarin glucuronide by capillary electrophoresis

Abstract

A method was developed for the determination of the in vitro production of 7-hydroxycoumarin glucuronide and β-glucuronidase activity in rabbit tissue homogenates (liver, kidney, heart, lung, spleen, large intestine and fat). Separation of 7-hydroxycoumarin (7-HC) and 7-hydroxycoumarin glucuronide (7-HCG) by capillary electrophoresis was carried out on a 27 cm untreated fused silica capillary using a 100 mM phosphate buffer, pH 7.0 at 17.5 kV, with detection at 320 nm. 7-HC and 7-HCG were separated within 4 min and could be determined in the same run. Rabbit tissues, containing uridine diphosphate (UDP) glucuronyl transferase (UDPGT), were homogenised in Tris–HCl, pH 7.4, and used for the production of 7-HCG by the reaction of 7-HC and UDP-glucuronic acid (UDPGA). The conversion of 7-HC to 7-HCG is catalysed by UDPGT and the reverse reaction by β-glucuronidase. A sample of the reaction mixture was removed and added to acetonitrile, centrifuged and analysed by capillary electrophoresis. For the reverse reaction (β-glucuronidase reaction), the rabbit tissue samples were homogenised in 100 mM acetate buffer, pH 4.3. To this 7-HCG was added and its metabolism to 7-HC and the decrease in 7-HCG content was determined after stopping the reaction with a β-glucuronidase inhibitor, protein precipitation and centrifugation. β-Glucuronidase activity was observed in all tissue types, but not all tissues displayed UDPGT activity. The highest UDPGT activity was detected in the liver, followed by the kidney. The limit of detection was 1 μg/ml for 7-HC, and 2 μg/ml for the glucuronide, with a linear detection range for both analytes from 0–100 μg/ml.