HIGHLY SENSITIVE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC DETERMINATION METHOD FOR A NEW ERYTHROMYCIN DERIVATIVE, EM523, AND ITS MAJOR METABOLITES IN HUMAN PLASMA AND URINE USING POSTCOLUMN TRIS(2,2'-BIPYRIDINE) RUTHENIUM(III) CHEMILUMINESCENCE DETECTION

A method for the simultaneous determination of de(N-methyl)-N-ethyl-8, 9-anhydroerythromycin A 6,9-hemiacetal (EM523, I) and its three metabo lites in human plasma and urine has been developed using high-performa nce liquid chromatography (HPLC) with chemiluminescence (CL) detection . Plasma and urine samples spiked with erythromycin as an internal sta ndard were extracted with a mixture of dichloromethane and diethyl eth er under alkaline conditions. The organic layer was evaporated under a stream of nitrogen gas. The reconstituted sample was injected into an HPLC apparatus and separated on an ODS column using a gradient elutio n method. The eluate was reacted on-line with a mixture of tris(2,2'-b ipyridine) ruthenium(II) and peroxodisulfate, and the generated CL int ensity was detected. Optimization of the CL reaction conditions result ed in a sensitive and stable CL intensity for the determination of I a nd its metabolites. The recovery of each compound from human plasma an d urine, and the sensitivity, linearity, accuracy and precision of the method were satisfactory. The lower limits of quantitation for each c ompound using 0.2 ml of plasma and 0.1 ml of urine were 1 and 10 ng/ml , respectively. This method has been used for the determination of I i n samples from clinical trials.