High-performance liquid chromatographic determination of a cysteine protease inhibitor and its ethyl ester in mouse serum and muscle by pre-column 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 microliters) and 300 pmol/g in muscle (20 mg), corresponding to ca. 0.5 pmol each in a 50-microliters 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.
Chao
W F
WF
Faculty of Pharmaceutical Sciences, Kyushu University 62, Fukuoka, Japan.
Kai
M
M
Ohkura
Y
Y
D000284:Administration, Oral; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D015853:Cysteine Proteinase Inhibitors / Q000008:administration & dosage / Q000032:analysis / Q000097:blood*; D005456:Fluorescent Dyes; D007930:Leucine / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000032:analysis / Q000097:blood; D051379:Mice; D008810:Mice, Inbred C57BL; D009132:Muscles / Q000032:analysis*; D013053:Spectrophotometry / Q000379:methods
D015853:Cysteine Proteinase Inhibitors; D005456:Fluorescent Dyes; D007930:Leucine; C108192:aloxistatin