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-2,6dimethyl-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 mu l mobile phase and 100 mu l of aliquots were injected to HPLC system. Chromatographic separation was accomplished on octadecyl column (250 mm x 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 (r(2) > 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. (c) 2005 Elsevier B.V. All rights reserved.