High-performance liquid chromatographic assay of fenflumizole and its demethyl metabolites in biological samples.

This paper describes sensitive, selective and precise methods for the assay of fenflumizole and its chromatographically verified demethyl and didemethyl metabolites in whole blood, isolated red blood cells, plasma, saliva, urine and tissue (skin and fat) from human subjects. Also conjugates of the two metabolites with glucuronic acid and sulphate were assayable. The compounds were quantitated by means of reversed-phase liquid chromatography after diethyl ether extraction, followed by fluorescence and/or electrochemical detection. The assay using fluorescence detection is quantitative down to ca. 150 pg/ml; with electrochemistry this limit was ca. 600 pg/ml and included the demethyl metabolites only. Proteinaceous materials show an extraction yield of 70-75%, whereas analytes in sample materials without proteins show yields of better than 95%. The precision at concentration levels of ca. 50 ng/ml for the parent compound and ca. 5 ng/ml for the metabolites is at most 6% (relative standard deviation) with both detection modes. The analytical procedures developed were applied after both single and repetitive administration of fenflumizole. The administration of 14C-labelled fenflumizole in the single-dose study revealed the presence in plasma and urine of as yet unknown metabolites. The in vivo retention time of 14C activity was substantially greater in the blood cells than in plasma. Measurements of 14C activity in excreta demonstrated that excretion via the faeces is the preferred route.