Use of post-column fluorescence derivatization to develop a liquid chromatographic assay for ranitidine and its metabolites in biological fluids

Ranitidine and its main metabolites, ranitidine N-oxide and ranitidine S-oxide, were determined in plasma and urine after separation using reversed-phase liquid chromatography. The mobile phase consisted of an initial isocratic step with 7:93 (v/v) acetonitrile-7.5 mM phosphate buffer (pH 6) for 8 min, followed by a linear gradient up to a 25:75 (v/v) mixture over 1 min. Detection was carried out by a post-column fluorimetric derivatization based on the reaction of the drugs with sodium hypochlorite, giving rise to primary amines that reacted with o-phthalaldehyde and 2-mercaptoethanol to form highly fluorescent products. The calibration graphs, based on peak area, were linear in the range 0.1-4 mu g/ml for all drugs. The detection limits were 30, 41 and 32 ng/ml (8.6, 12.5 and 9.1 pmol) for ranitidine S-oxide, ranitidine N-oxide and ranitidine, respectively. Chromatographic profiles obtained for plasma and urine samples showed no interference from endogenous compounds.