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.

A method for the rapid determination of 7-hydroxycoumarin, based on separation by capillary electrophoresis (CE), with UV absorbance detection at 210 nm, was applied to a study of the metabolism of coumarin in human liver microsomes. The linear detection range for 7-hydroxycoumarin was 0–50 μg/ml, while the limit of detection was 1 μg/ml. Human liver microsomes from 5 patients were used in metabolic studies and the profiles clearly indicated that there is inter-individual variability in coumarin 7-hydroxylase activity. The CE method allowed the investigation of NADP+NADPH cofactor reactions involved in coumarin metabolism. The CE method was also compared to a liquid chromatographic method for the analysis of the metabolite and it was found that the results did not differ but the CE method was more rapid and needed no sample preparation. The very short time between sampling and analysis makes the method suitable for automated sample transfer and measurement.

The antibody-antigen interaction is characterised by high affinity and high specificity, which makes it an excellent candidate as an analytical tool for selective and sensitive determinations. This biochemical interaction is being increasingly detected by solid-state physical transducers, in devices which are termed 'biosensors'. The structure of the antibody molecule is central to its binding function. In addition, several techniques are available by which antibodies may be produced. An appreciation of both of these areas is essential in developing an immunosensor. The choice of the appropriate transducer is also important, and this short article also highlights some recent applications involving the use of electrochemical, optical, surface acoustic wave and semiconductor transducers.

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 μg/ml while the limit of quantitation was 1 μg/ml. An internal standard, 3-(α-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.

An immunosensor based on a competitive electrochemical immunoassay was developed for the determination of 7-hydroxycoumarin. The antibody-based biosensor employed horseradish peroxidase-labelled anti-7-hydroxycoumarin, with the enzyme catalysed reaction involving the reduction of hydrogen peroxide in the presence of a mediator (hydroquinone). Sensor preparation involved immobilisation of the protein (ovalbumin/thyroglobulin)-bound 7-hydroxycoumarin antigen within a Nafion film on a glassy carbon surface. The electrode was then incubated in a solution containing both the enzyme-labelled anti-7-hydroxycoumarin and increasing concentrations of the free 7-hydroxycoumarin analyte. Competition between the free and immobilised forms of the antigen allows for the quantitation of free 7-hydroxycoumarin in solution. Injections of the enzyme substrate, hydrogen peroxide, resulted in an increase in the steady-state cathodic current (−0.03 V vs. saturated calomel reference electrode). This current output was inversely proportional to the concentration of analyte in solution. Free 7-hydroxycoumarin in solution was determined between 0 and 1 mM, with a detection limit of 24 μM.

7-Hydroxycoumarin (7-HC) was chemically conjugated by diazo coupling to carrier proteins such as bovine serum albumin (BSA), thyroglobulin and ovalbumin. These conjugates were characterised by sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC). Rabbits were immunised using the 7-HC-BSA conjugate. The highest antibody titre achieved was 1:10000, as determined by competitive enzyme-linked immunosorbent assay (ELISA). The resulting antibodies were purified by ammonium sulphate precipitation, followed by protein A affinity chromatography. Their purity was assessed by SDS-PAGE and HPLC. These antibodies have been used in the development of a competitive ELISA, an amperometric biosensor and an electrochemical immunoassay. Both the ELISA and amperometric biosensor have been successfully applied to the analysis of 7-HC and its glucuronide conjugate in human urine samples. Each of these antibody-based methods provides a novel approach to the analysis of the main metabolites of coumarin.