Quantification of l-3-(3-hydroxy-4-pivaloyloxyphenyl) alanine (NB-355) by high-performance liquid chromatography using o-phthalaldehyde/N-acetyl- l-cysteine derivatization

Abstract

A new and rapid high-performance liquid chromatographic assay has been developed for the determination of l-3-(3-hydroxy-4-pivaloyloxyphenyl) alanine (NB-355, I), a novel prodrug of l-DOPA. The method involves precolumn derivatization of the drug in biological samples with o-phthalaldehyde (OPA) and N-acetyl- l-cysteine (NAC) in a triethanolamine buffer (pH 8.0), giving a fluorescent compound that is stable for 2 h at 4°C. Use of an internal standard improved the assay in accuracy and reliability. A programmable injector allowed automatic derivatization of large numbers of samples. Chromatographic separation was performed on a reversed-phase column (Capcell Pak C 18) in which the silica gel was coated with silicone polymer. The peaks corresponding to compound I and the internal standard were eluted within 16 min with a mobile phase of acetonitrile-phosphate buffer (pH 7.1). The reliable limit of quantification was 0.5 pmol per injection (0.05 μg equivalents of l-DOPA per ml in plasma). The method was successfully applied for the measurements of dog plasma concentrations after oral dosing of compound I.