ANALYSIS OF THE GLUCURONIDATION OF 7-HYDROXYCOUMARIN BY HPLC

The in-vitro metabolism of 7-hydroxycoumarin to 7-hydroxycoumarin-gluc uronide was investigated in bovine liver homogenate. A metabolic react ion mixture was prepared that included a crude preparation of uridine diphosphate (UDP) glucuronyl transferase, 7-hydroxycoumarin and UDP-gl ucuronic acid. A HPLC method was developed to separate coumarin, 7-hyd roxycoumarin, 7-hydroxycoumarin-glucuronide and an internal standard, 4-hydroxycoumarin. Samples were separated by reverse-phase HPLC, on a C18 column, with a 1 mi min(-1) gradient elution with UV detection at 320 nm. The limit of quantification of the method, for 7-hydroxycoumar in-glucuronide, was 1.47 mu M, and the linear range was from 0-295.7 m u M. Concentrations of 7-hydroxycoumarin-glucuronide produced were cal culated from a plot of 7-hydroxycoumarin-glucuronide concentration ver sus the mean absorbance ratio (n = 4) (7-hydroxycoumarin-glucuronide a bsorbance/4-hydroxycoumarin absorbance). It was possible to monitor th e decrease in the 7-hydroxycoumarin content as it was metabolised as w ell as the increase in 7-hydroxycoumarin-glucuronide as it was produce d enzymatically. The identity of the compound produced was confirmed b y photodiode array spectral analysis. A plot of time versus 7-hydroxyc oumarin-glucuronide produced indicates that the metabolism is linear f or the first 90 min and reached a plateau at 150 min. The rate of reac tion in the first 90 min was 2.96 +/- 0.06 (RSD 1.7%, n = 3) nmol of 7 -hydroxycoumarin-glucuronide produced per minute per milligram of prot ein. After 150 min 0.34 +/- 0.005 mM (RSD 1.4%) 7-hydroxycoumarin-gluc uronide was produced, from 0.77 mM 7-hydroxycoumarin introduced into t he reaction mixture and 58.0% +/- 5.3% (or 0.44 +/- 0.02 mM) of the 7- hydroxycoumarin remained. These results show that it is possible to mo nitor the production of the phase II metabolite of coumarin with minim al sample clean-up and without the need for deconjugation of the glucu ronide moiety. The method was very reliable and applicable for the dir ect determination of 7-hydroxycoumarin-glucuronide in an in-vitro meta bolic assay.