Determination of sq 27,519, the active phosphinic acid—carboxylic acid of the prodrug sq 28,555, in human serum by capillary gas chromatography with nitrogen—phosphorus detection after a two-step derivatization

Abstract

A method for the determination of SQ 27,519 (II), the active phosphinic acid—carboxylic acid of the prodrug SQ 28,555 (I), in human serum is presented. Compounds I and II are simultaneously extracted from acidified serum into ethyl acetate, and II is back-extracted into aqueous sodium bicarbonate. Compound I, in ethyl acetate, can be subsequently hydrolyzed and measured as II. The two acidic groups of II are selectively esterified, first by methylation of the carboxylic acid with methanolic hydrochloric acid and then by formation of the hexafluoroisopropyl ester of the phosphinic acid. The resulting product is measured by splitless-injection capillary gas chromatography with nitrogen—phosphorus detection. Linear standard curves were obtained for II with a detection limit of less than 10 ng/ml of serum. The method was successfully applied to the analysis of serum samples obtained from normal individuals after administration of I. In an ascending-dose study involving several human subjects the serum levels of II ranged from less than 10 to 7000 ng/ml of serum.