Direct determination of 7-hydroxycoumarin and 7-hydroxycoumarin-glucuronide in urine by using capillary electrophoresis

Abstract

A method has been developed for the direct determination of 7-hydroxycoumarin (7HC) and 7-hydroxycoumaringlucuronide (7HCG) in urine without sample clean-up. Separation was carried out in 90% 100 mmol l^–1 phosphate buffer, 11 mmol l^–1 deoxycholic acid (sodium salt) and 10% acetonitrile, on a 47 cm uncoated silica capillary at 20 kV with detection of the analytes at 320 nm. The linear detection range for concentration versus peak area for the assay is from 0 to 100 µg ml^–1 for both analytes, with a limit of quantitation of 2 µg ml^–1 for 7HC and 5 µg ml^–1 for 7HCG in urine. Inter-and intra-assay results showed s_r values in peak areas of between 0.5 and 13%. The method was applied to the direct determination of 7HC and the glucuronide conjugate in urine from two volunteers administered with 250 mg of coumarin. The samples were also analysed by another CE method and by using HPLC. There was no statistical difference between the results determined by each of the methods. Up to 83% of the coumarin administered was excreted as 7HC or 7HCG. The majority of the 7HC excreted was in the glucuronide form (98%) with 2% occurring as free 7HC.