Analysis of Erythromycin A and Its Metabolites in Biological Samples by Liquid Chromatography with Post-Column Ion-Pair Extraction

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Abstract
The development of a selective and sensitive method for analysis of erythromycin 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 × 4.6 mm I.D. poly(styrene-divinylbenzene) (PLRP-S 8 μm, 1000 Å) column at 70°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 on-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 its potential metabolites in plasma and urine were linear over the concentration range studied. The detection limits were 12.5 ng/ml plasma and 50 ng/ml urine. The method allows the detection of all the erythromycin metabolites in human plasma and urine.

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