DETERMINATION OF ALPIDEM, AN IMIDAZOPYRIDINE ANXIOLYTIC, AND ITS METABOLITES BY COLUMN-SWITCHING HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH FLUORESCENCE DETECTION

Alpidem, l)-N,N-dipropylimidazo[1,2-a]pyridine-3-acetamide, is an anxi olytic imidazopyridine that undergoes a first-pass elimination after o ral administration to humans; it is actively metabolized and three cir culating metabolites have been identified in plasma due to N-dealkylat ion, oxidation or a combination of both processes. For the determinati on of the unchanged drug and its metabolites in human plasma, a column -switching HPLC method was developed. The method, based on solid-phase extraction (performed on-line), involves the automatic injection of p lasma samples (200 mu ul) on to a precolumn filled with C-18 material, clean-up of the sample with water in order to remove protein and salt s and transfer of the analytes to the analytical column (after valve s witching) by means of the mobile phase. All the processes were perform ed in the presence of an internal standard, a compound chemically rela ted to alpidem. During the analytical chromatography, the precolumn wa s flushed with different solvents and after regeneration with water, i t was ready for further injections. The analytical column was a C-8 ty pe and the mobile phase was acetonitrile-methanol-phosphate buffer sol ution (45:15:45, v/v/v) at a flow-rate of 1.5 mi min(-1). The column w as connected to a fluorimetric detector operating at excitation and em ission wavelengths of 255 and 423 nm, respectively. The limits of quan titation of alpidem and three metabolites were 2.5 and 1.5 ng ml(-1), respectively, in human plasma.