Simultaneous determination of coumarin, 7-hydroxycoumarin and 7-hydroxycoumarin glucuronide in human serum and plasma by high-performance liquid chromatography.

A HPLC method was developed for the determination of the metabolites of coumarin and 7-hydroxycoumarin in plasma and serum. Separation was based on gradient elution of 7-hydroxycoumarin glucuronide, 7-hydroxycoumarin, coumarin and finally 4-hydroxycoumarin (which is used as an internal standard). Standards, prepared in plasma or serum, and samples were treated with trichloroacetic acid, mixed and centrifuged. The supernatant was removed and analyzed by reversed-phase high-performance liquid chromatography on a C18 column. The limit of detection was 50 ng/ml for 7-hydroxycoumarin and 200 ng/ml for coumarin and 7-hydroxycoumarin glucuronide. The linear range was 0.5-100 micrograms/ml for each of the analytes. The percentage relative standard deviation about the mean measured concentrations were all below 10%. There was no statistical difference between the standard curves prepared in plasma or serum. The method developed was applied to the determination of each of the three compounds in serum, after the administration of 7-hydroxycoumarin, and in plasma after the administration of coumarin. The concentrations of total 7-hydroxycoumarin in the serum samples were also determined by another HPLC method and the results were compared. There was no statistical difference between the results determined.

Analysis of the glucuronidation of 7-hydroxycoumarin by HPLC.

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 uridine diphosphate (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 a C18 column, with a 1 ml min-1 gradient elution with UV detection at 320 nm. The limit of quantification of the method, for 7-hydroxycoumarin-glucuronide, was 1.47 microM, and the linear range was from 0-295.7 microM. 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.

Interspecies differences in coumarin metabolism in liver microsomes examined by capillary electrophoresis.

1. A simple, rapid method was developed for studying xenobiotic metabolism by cytochrome P450 in liver microsome preparations. Capillary electrophoresis was used to separate the metabolite from the metabolic mixture. 2. Coumarin is metabolized to 7-hydroxycoumarin by a cytochrome P450 isoenzyme. Human, bovine, gerbil, mouse (Schofield, CO1), rat, rabbit, porcine, and cynomologus monkey microsomal preparations were investigated for coumarin metabolism by determining the content of 7-hydroxycoumarin present after metabolism. 3. Separation of 7-hydroxycoumarin from the reaction mixture was carried out in 50 mM phosphate buffer, pH 6.8, on a fused silica capillary at 25 degrees C and 15 kV. The metabolic matrix consisted of an NADPH regeneration system, 205.5 mu M coumarin, and the microsomal preparation. Standard curves were prepared in the microsomal preparation and the limit of quantification was 6.17 mu M, with a linear range from 0 to 308.5 mu M. 4. The reaction was initiated by the addition of the microsomes. An aliquot of the reaction mixture was removed at specific timed intervals over 2 h and injected directly onto a capillary electrophoresis column and the concentration of 7-hydroxycoumarin determined. The metabolism of coumarin to 7-hydroxycoumarin is greatest in human and monkey microsomes.

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

A method has been developed for the direct determination of 7-hydroxycoumarin (7HC) and 7-hydroxycoumarin-glucuronide (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 micrograms ml-1 for both analytes, with a limit of quantitation of 2 micrograms ml-1 for 7HC and 5 micrograms ml-1 for 7HCG in urine. Inter- and intra-assay results showed sr 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 statical 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.

The use of capillary electrophoresis for studying the in vitro glucuronidation of 7-hydroxycoumarin.

An assay utilizing CE, has been developed for studying the in vitro metabolism of 7-hydroxycoumarin to 7-hydroxycoumarin-glucuronide. A reaction mixture containing a crude preparation of bovine uridine diphosphate (UDP) glucuronyl transferase (UDPGT) and the substrates uridine diphosphate glucuronic acid (UDPGA) and 7-hydroxycoumarin was prepared. Aliquots were removed from the reaction mixture at specific time intervals and analyzed on a 57-cm untreated silica capillary for the presence of 7-hydroxycoumarin-glucuronide. Samples were electrophoresed in a 100 mM phosphate/11 mM deoxycholic acid (sodium salt)/acetonitrile electrolyte buffer, pH 7.0, at 30 kV, with detection at 320 nm. The method allowed the determination of 7-hydroxycoumarin-glucuronide without sample cleanup, with a limit of detection of 2 micrograms/mL, and a linear detection range of 0 microgram/mL to 100 micrograms/mL. The 7-hydroxycoumarin-glucuronide, concentrations were calculated from calibration curves of standards prepared in enzyme solution. The initial rate of production of 7-hydroxycoumarin-glucuronide, in the first 70 min, was 3.1 +/- 0.13 nmol/mL/min/mg protein. The method was fast and reliable with percentage relative standard deviations for concentration of 7-hydroxycoumarin-glucuronide, over time, of less than 10%.

Determination of free and total 7-hydroxycoumarin in urine and serum by capillary electrophoresis.

A new method for the rapid determination of 7-hydroxycoumarin, the predominant metabolite of coumarin in humans, was developed for analysis in urine and serum, based on separation by capillary electrophoresis, with UV detection at 210 nm. The linear detection range for 7-hydroxycoumarin was 0-50 micrograms/ml while the limit of quantitation was 1 microgram/ml. An internal standard, 3-(alpha-acetonylbenzyl)-4-hydroxycoumarin, was utilised for the determination of free 7-hydroxycoumarin, but it was found not to be suitable in the analysis of total 7-hydroxycoumarin present. Urine from two volunteers, who had been administered coumarin, was analysed by both capillary electrophoresis and by HPLC. The results from the two methods were compared and contrasted. The CE method was found to decrease the analysis time in comparison to HPLC analysis, with results available after 1.5 min as compared to 12 min with HPLC. There was no statistical difference between the results determined by either method.