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Titolo:
ANALYSIS OF THE GLUCURONIDATION OF 7-HYDROXYCOUMARIN BY HPLC
Autore:
KILLARD AJ; OKENNEDY R; BOGAN DP;
Indirizzi:
DUBLIN CITY UNIV,SCH BIOL SCI DUBLIN 9 IRELAND DUBLIN CITY UNIV,SCH BIOL SCI DUBLIN 9 IRELAND
Titolo Testata:
Journal of pharmaceutical and biomedical analysis
fascicolo: 11, volume: 14, anno: 1996,
pagine: 1585 - 1590
SICI:
0731-7085(1996)14:11<1585:AOTGO7>2.0.ZU;2-C
Fonte:
ISI
Lingua:
ENG
Soggetto:
COUMARIN 7-HYDROXYLATION; INTERINDIVIDUAL VARIABILITY; CAPILLARY ELECTROPHORESIS; METABOLISM;
Keywords:
HPLC; 7-HYDROXYCOUMARIN; 7-HYDROXYCOUMARIN-GLUCURONIDE; URIDINE DIPHOSPHATE-GLUCURONYL TRANSFERASE;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
20
Recensione:
Indirizzi per estratti:
Citazione:
A.J. Killard et al., "ANALYSIS OF THE GLUCURONIDATION OF 7-HYDROXYCOUMARIN BY HPLC", Journal of pharmaceutical and biomedical analysis, 14(11), 1996, pp. 1585-1590

Abstract

The in-vitro metabolism of 7-hydroxycoumarin to 7-hydroxycoumarin-glucuronide was investigated in bovine liver homogenate. A metabolic reaction mixture was prepared that included a crude preparation of uridinediphosphate (UDP) glucuronyl transferase, 7-hydroxycoumarin and UDP-glucuronic acid. A HPLC method was developed to separate coumarin, 7-hydroxycoumarin, 7-hydroxycoumarin-glucuronide and an internal standard,4-hydroxycoumarin. Samples were separated by reverse-phase HPLC, on aC18 column, with a 1 mi min(-1) gradient elution with UV detection at320 nm. The limit of quantification of the method, for 7-hydroxycoumarin-glucuronide, was 1.47 mu M, and the linear range was from 0-295.7 mu M. Concentrations of 7-hydroxycoumarin-glucuronide produced were calculated from a plot of 7-hydroxycoumarin-glucuronide concentration versus the mean absorbance ratio (n = 4) (7-hydroxycoumarin-glucuronide absorbance/4-hydroxycoumarin absorbance). It was possible to monitor the decrease in the 7-hydroxycoumarin content as it was metabolised as well as the increase in 7-hydroxycoumarin-glucuronide as it was produced enzymatically. The identity of the compound produced was confirmed by photodiode array spectral analysis. A plot of time versus 7-hydroxycoumarin-glucuronide produced indicates that the metabolism is linear for the first 90 min and reached a plateau at 150 min. The rate of reaction 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 protein. After 150 min 0.34 +/- 0.005 mM (RSD 1.4%) 7-hydroxycoumarin-glucuronide was produced, from 0.77 mM 7-hydroxycoumarin introduced into the 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 monitor the production of the phase II metabolite of coumarin with minimal sample clean-up and without the need for deconjugation of the glucuronide moiety. The method was very reliable and applicable for the direct determination of 7-hydroxycoumarin-glucuronide in an in-vitro metabolic assay.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 13/07/20 alle ore 04:58:48