HPLC Assay for Basic Amine Drug in Plasma Using a Silica Gel Column and an Aqueous Mobile Phase–Application in a Pilot Bioavailability Study of Chlorpheniramine Controlled-Release Dosage Form

Abstract

A high performance liquid chromatographic (HPLC) method that involves the use of a silica gel column and an aqueous mobile phase for simultaneous separation of chlorpheniramine, pseudoephedrine and terfenadine in plasma is presented. Alkalized samples are cleaned by extraction with n-hexane, and the extraction is followed by evaporating the solvent and reconstituting the residue in a small amount of mobile phase. An aliquot of this solution is analyzed by a HPLC system with a silica gel column, an aqueous mobile phase containing 55% CH₃CN and 45% (NH₄)H₂PO4(pH 4.0), and UV detection at 210 nm. The low detection limits of the method in plasma are 1 ng, 4 ng and 0.5 ng for chlorpheniramine, pseudoephedrine and terfenadine, respectively. In this study, terfenadine acts as an internal standard. The coefficient of variance on the results of intraday and interday precision and the accuracy on control samples of chlorpheniramine and pseudoephedrine were all within 10%. We have used this method successfully in a pilot bioavailability study of a newly developed controlled-release formulation.