Spectrofluorimetric method for the quantification of 7-hydroxycoumarin in urine and plasma using both extracted and unextracted samples

Abstract

A sensitive spectrofluorimetric assay has been developed for the determination of 7-hydroxycoumarin (7-OHC) in urine and plasma. Assay systems for urine and plasma were developed and compared with regard to linearity, accuracy, precision, limit of quantification, percentage recovery and assay time. The amount of 7-OHC conjugated to glucuronide and excreted in the urine, was determined following treatment with β-glucuronidase (5000 U ml^–1; 1 U = 16.67 nkat) for 30 min at 37 °C. This allows the urinary concentration of free, total and conjugated 7-OHC to be determined. Samples were extracted with diethyl ether and a suitable aliquot of reconstituted extract diluted in 0.1 mol l^–1 phosphate buffered saline (PBS), pH 10.0, and transferred to a 96 well microtitre plate. The 7-OHC concentration was also determined without prior solvent extraction. Aliquots (20 µl) of urine and plasma samples were transferred to a 96 well microtitre plate and diluted to 200 µl with PBS, pH 10.0. The fluorescence intensity was determined at excitation and emission wavelengths of 370 and 450 nm, respectively, for both extracted and unextracted samples. Linear ranges of 7-OHC in urine and plasma, using either method, were found to be 0.5–10 and 10–100 µg ml^–1. Inter- and intra-day precision studies, using both methods, demonstrated relative standard deviations of below 10% across the linear range. The limit of quantification of 7-OHC in urine and plasma was 0.5 µg ml^–1 using both methods. Both methods have been used successfully to determine the concentration of 7-OHC excreted in the urine of patients. The percentage of the dose recovered as 7-OHC over a 24 h period was 92–98%, using unextracted and extracted samples, respectively, with 98% of this recovered as the glucuronide conjugate. The two methods are a significant improvement on a previously described method and provide an alternative to high-performance liquid chromatography.