A simple, rapid, and sensitive validated spectrophotometric method was developed for the determination of certain macrolide antibiotics namely, erythromycin (I), azithromycin dihydrate (II), clarithromycin (III), and roxithromycin (IV) in bulk powders, pharmaceutical formulations, and spiked biological fluids. The proposed method is based on the formation of a binary complex between each of the studied drugs and eosin Y in aqueous buffered medium. Under the optimum conditions, the binary complexes showed absorption maxima at 542-544 nm. The absorbance of the binary complexes obeyed Beer's law over the concentration range of 1-10 micro/g/mL for II, 2-20 microg/mL for I and IV, and 3-30 microg/mL for III. The mean percentage recoveries were 100.04 +/- 0.83, 99.98 +/- 0.80, 100.17 +/- 0.91, and 99.55 +/- 0.91, with minimum detectable molarities of 2 x 10(-7) for I and II, 4 x 10(-7) for III, and 3 x 10(-7) for IV. The different experimental parameters affecting the development and stability of the colors were studied and optimized. The proposed method was successfully applied to the analysis of the cited drugs in some pharmaceutical formulations. The results obtained were in good agreement with those obtained using the reference methods. The proposed method was further applied to spiked human urine and plasma. A proposal of the reaction pathway is suggested.

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