Direct Fluorescence and Spectrophotometric-based Detection of Azithromycin Using Fluorescein Isothiocyanate: Method Development and Comparative Analysis

New methods for the analysis of azithromycin (AZM) have been devised, employing direct fluorescence and spectrophotometric techniques. This research focused on the development and validation of these methods, especially based on the formation of an ion-pair complex with fluorescein isothiocyanate (FITC). The resulting complex enhanced the fluorescence intensity at a wavelength of 520nm and the absorbance at 480nm. The impact of different solvent compositions was investigated, with methanol and a 1:1 mixture of methanol and Milli-Q proving to be the most sensitive. The optimal FITC concentration was determined as 50µg/ml, and the molar ratio of the AZM-FITC ion-pair complex was determined to be 1:1. Notably, an instantaneous increase in fluorescence intensity was observed, eliminating the need for incubation. The developed methods were validated for sensitivity, accuracy, and precision, with the fluorescence-based method demonstrating superior sensitivity and precision compared to the spectrophotometric method. Under optimum conditions, the fluorescence-based method achieved a linear range of 0-31.25µg/ml with a limit of detection (LOD) of 0.41µg/ml and a limit of quantification (LOQ) of 1.23µg/ml., In contrast, the spectrophotometric method had an LOD of 0.82µg/ml and an LOQ of 2.49µg/ml. Additionally, the fluorescence-based method exhibited a narrower recovery range in artificial urine samples, suggesting higher precision in complex matrices. Overall, these methods offer rapid and sensitive AZM analysis with potential applications in diverse matrices such as biological samples post-extraction.