HPLC method for analysis of a new 1,4-dihydropyridine: Application to pharmacokinetic study in rabbit

A high sensitive HPLC assay for plasma analysis of a new 1,4-dihydropyridine (nitrimidodipine) was developed to support the subsequent preclinical development of the compound. To 1 ml of rabbit plasma was added internal standard (3-(4-nitrooxy butyl)-5-ethyl-1,4-dihydro-2,6-dimethyl-4-(1-methyl-5-nitro-2-imidazolyl)-3,5-pyridine dicarboxylate) and 0.5 ml of 1 M HCl. The plasma was extracted using 5 ml ethyl acetate which evaporated under gentle stream of nitrogen. The residue was reconstituted in 200 μl mobile phase and 100 μl of aliquots were injected to HPLC system. Chromatographic separation was accomplished on octadecyl column (250 mm × 4.6 mm) using a mobile phase consisting of acetonitrile–water (45:55, v/v). The method was sensitive to 2.5 ng/ml in plasma (LOD), acceptable within- and between day reproducibility and a linearity (r2 > 0.9957) over a concentration range from 5 to 400 ng/ml. The mean extraction efficacy was 90.6% and no interfering peaks of the blank plasma chromatograms were observed. By using the above procedure, a simple, sensitive and convenient HPLC assay for determination, stability evaluation and pharmacokinetic study of nitrimidodipine was developed.