High-performance liquid chromatographic method for the determination of eclanamine and its N-desmethyl and N,N-didesmethyl metabolites in urine

A high-performance liquid chromatographic method has been defined for the determination of eclanamine (free base of eclanamine maleate) and two of its metabolites, N-desmethyleclanamine and N,N-didesmethyleclanamine in urine. The method employs 10-ml urine samples, has a linear range from 5 to 500 ng/ml for the three compounds, and has a detection limit of 0.5 ng/ml for each compound. Sample preparation uses a cyanopropylsilane extraction column with washes of water, acetonitrile-water (30:70, v/v), and acetonitrile, and elution with 2% trifluoroacetic acid in acetonitrile. The eluate is evaporated to dryness, the residue dissolved in 1.0 ml acetonitrile-water (10:90, v/v) and 100 microliter are injected onto a Supelcosil LC-CN column. Eclanamine and its metabolites are eluted with an acetonitrile-water (35:65, v/v) eluent containing 0.01 M triethylamine and adjusted to pH 7.0 with phosphoric acid. The method has been validated by preparing and analyzing a series of fortified urines (range 2-500 ng/ml for each compound) on four separate days. Good linearity, precision, reproducibility, and specificity were obtained. Certification of the analytical method was accomplished by analyzing urine specimens collected from one volunteer administered a single oral dose of 45 mg eclanamine maleate. The data suggest that the metabolites of eclanamine have long elimination half-lives with levels still quantifiable in the 72-96 h collection interval.