AUTOMATED-DETERMINATION OF VERAPAMIL AND NORVERAPAMIL IN HUMAN PLASMAWITH ONLINE COUPLING OF DIALYSIS TO HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY AND FLUOROMETRIC DETECTION

A fully automated method for the simultaneous determination of verapam il and its main metabolite norverapamil in human plasma is described. This method is based on on-line sample preparation using dialysis foll owed by clean-up and enrichment of the dialysate on a precolumn and su bsequent HPLC analysis with fluorometric detection. All sample handlin g operations were performed automatically by a sample processor equipp ed with a robotic arm (ASTED system). The plasma samples were dialysed on a cellulose acetate membrane (cut-off: 15 kD) and the dialysate wa s purified and enriched on a short pre-column filled with cyanopropyl silica. Before starting dialysis, this trace enrichment column (TEC) w as first conditioned with the HPLC mobile phase and then with pH 3.0 a cetate buffer. 370 mu l of plasma sample spiked with the internal stan dard (gallopamil) were dialysed in the static-pulsed mode. The solutio n at the donor side was pH 3.0 acetate buffer containing Triton X-100 while the acceptor solution was made of the same acetate buffer. When dialysis was discontinued, the analytes were desorbed from the TEC by the HPLC mobile phase and transferred to the C-18 analytical column by means of a switching valve. This mobile phase consisted of a mixture of acetonitrile, pH 3.0 acetate buffer and 2-aminoheptane. The influen ce of different parameters of the dialysis process on the recovery of verapamil and norverapamil has been studied. The effect of the volume, the aspirating and dispensing flow-rates of the dialysis solution has been investigated. The recoveries of verapamil and norverapamil in pl asma were close to 75% and the limits of quantification were 5 ng/ml f or both analytes. The method was found to be linear in the concentrati on range from 5 to 500 ng/ml (r(2): 0.9996 for both analytes). The int ra-day and inter-day reproducilities at a concentration Of 100 ng/ml w ere 2.3% and 5.6% for verapamil and 1.7% and 5.1% for norverapamil, re spectively.