ANALYSIS OF ERYTHROMYCIN-A AND ITS METABOLITES IN BIOLOGICAL SAMPLES BY LIQUID-CHROMATOGRAPHY WITH POSTCOLUMN ION-PAIR EXTRACTION

The development of a selective and sensitive method for analysis of er ythromycin A (EA) and its metabolites in biological fluids (urine and plasma) is reported here. A mobile phase consisting of acetonitrile - 2-methyl-2-propanol - 0.2 M phosphate buffer pH 9.0 - water (3:19:5:73 , v/v) at 1.5 ml/min enables the separation of the main component (EA) from all of its potential metabolites on a 250 x 4.6 mm I.D. poly(sty rene-divinylbenzene) (PLRP-S 8 mu m, 1000 Angstrom) column at 70 degre es C. To improve the sensitivity of the method, a post-column ion-pair extraction with the strongly fluorescent 9, 10-dimethoxy-anthracene-2 -sulphonate was used. The ion-pairs were extracted in chloroform for o n-line fluorescence detection. Analytical recoveries for erythromycin and its metabolites after extraction of the plasma with tertiary butyl methyl ether were better than 90%. The calibration curves of EA and i ts potential metabolites in plasma and urine were linear over the conc entration range studied. The detection limits were 12.5 ng/ml plasma a nd 50 ng/ml urine. The method allows the detection of all the erythrom ycin metabolites in human plasma and urine.