HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC DETERMINATION OF A CYSTEINE PROTEASE INHIBITOR AND ITS ETHYL-ESTER IN MOUSE SERUM AND MUSCLE BY PRECOLUMN FLUORESCENCE DERIVATIZATION

A simple and sensitive high-performance liquid chromatographic method has been developed for the determination of a cysteine protease inhibitor (E-64-c) and its ethyl ester in mouse serum and muscle samples. After deproteinization with acetone, E-64-c is converted into a fluorescent derivative by reaction with 3-bromomethyl-6,7-dimethoxy-1-methyl-2 (1H)-quinoxalinone. The derivative is separated on a reversed-phase column by isocratic elution of aqueous acetonitrile and monitored fluorimetrically. The ethyl ester is hydrolysed to E-64-c by carboxyl esterase and then derivatized in the same way as E-64-c. The limits of detection, at a signal-to-noise ratio of 3, of E-64-c and the ester are 500 pmol/ml in serum (10 μl) and 300 pmol/g in muscle (20 mg), corresponding to ca. 0.5 pmol each in a 50-μl injection volume. The method allows the determination of E-64-c and the ethyl ester in mouse serum and muscle after oral administration of these compounds. © 1990.