Sensitive and specific high-performance liquid chromatographic assay for the quantification of sulforidazine and two diastereomeric sulforidazine-5-sulfoxide metabolites in plasma.

A sensitive and specific procedure using high-performance liquid chromatography for the quantification of sulforidazine and two diastereomeric sulforidazine-5-sulfoxide metabolites in plasma was developed. Sulforidazine was first extracted from basified plasma using a mixture of pentane and 2-propanol. Sulforidazine-5-sulfoxide metabolites were then extracted from the same basified plasma using a second solvent mixture consisting of methylene chloride, pentane and 2-propanol. Each organic extract was subsequently back-extracted separately with 0.1 M hydrochloric acid, basified and re-extracted with the original solvent mixtures. In order to avoid interferences due to hydroxylated metabolites co-eluting with sulforidazine-5-sulfoxides in the more polar extract, this extract was derivatized with N-methyl-(tert.-butyldimethylsilyl) trifluoroacetamide. The two extracts were separately chromatographed on a narrow-bore nitrile column using ultraviolet detection. The quantification limits for sulforidazine and two diastereomeric sulforidazine-5-sulfoxide metabolites were 1.0 ng/ml with mean intra-assay coefficients of variation less than 10%. These methods were applied to the analysis of plasma from a dog following the administration of a single oral dose (25 mg of the base) of sulforidazine hydrochloride.