Simultaneous high-performance liquid chromatography assay of two metabolites of 6-chloro-2-pyridylmethyl nitrate, a new antianginal drug, and its application to a pharmacokinetic study in rats.

Abstract

An HPLC technique has been developed for the simultaneous determination of 6-chloro-2-pyridine methanol (5) and 6-chloro-2-pyridinecarboxylic acid (3), two metabolites of 6-chloro-2-pyridylmethyl nitrate, a new antianginal drug, in rat plasma. The plasma sample was placed on a Bond Elut C18 column and the compounds were eluted with 250 microL of the mobile phase. A 50-150-microL aliquot was injected onto the HPLC column. No interfering substances were observed in the plasma of a normal rat. The calibration curves for both 5 and 3 were linear from 0.1 to 50 micrograms/mL. However, the quantitative detection of the two additional metabolites, N-(6-chloro-2-pyridylcarbonyl)-glycine (1) and N-acetyl-S-(6-chloro-2-pyridylmethyl)-L-cysteine (2) was not satisfactory. The metabolic pathway of 6-chloro-2-pyridylmethyl nitrate in vivo was studied in male rats which were dosed separately with 6-chloro-2-pyridylmethyl nitrate, 5, and 3. After intravenous injection of 6-chloro-2-pyridylmethyl nitrate, the unchanged drug was determined by a GC method that was also developed by us and reported in another paper. 6-Chloro-2-pyridylmethyl nitrate was rapidly metabolized, and the metabolites were detected as early as 1 min after 6-chloro-2-pyridylmethyl nitrate administration. The parent compound then declined rapidly and 3 formation was confirmed in the rat plasma. 6-Chloro-2-pyridylmethyl nitrate is extensively metabolized to 3 via 5 formation, and 3 is the main metabolite in rat plasma.