Quantitation of promethazine and metabolites in urine samples using on-line solid-phase extraction and column-switching

A chromatographic method for the quantitation of promethazine (PMZ) and its three metabolites in urine employing on-line solid-phase extraction and co lumn-switching has been developed. The column-switching system described he re uses an extraction column for the purification of PMZ and its metabolite s from a urine matrix, The extraneous matrix interference was removed by fl ushing the extraction column with a gradient elution. The analytes of inter est were then eluted onto an analytical column for further chromatographic separation using a mobile phase of greater solvent strength. This method is specific and sensitive with a range of 3.75-1400 ng/ml for PMZ and 2.5-140 0 ng/ml for the metabolites promethazine sulfoxide, monodesmethyl promethaz ine sulfoxide and monodesmethyl promethazine. The lower limits of quantitat ion (LLOQ) were 3.75 ng/ml with less than 6.2% CV for PMZ and 2.50 ng/ml wi th less than 11.5% CV for metabolites based on a signal-to-noise ratio of 1 0:1 or greater. The accuracy and precision were within +/- 11.8% in bias an d not greater than 5.5% CV in intra- and inter-assay precision for PMZ and metabolites. Method robustness was investigated using a Plackett-Bunnan exp erimental design. The applicability of the analytical method for pharmacoki netic studies in humans is illustrated. (C) 2001 Elsevier Science B.V. All rights reserved.