Eosin Y Complexation Enables Spectrophotometric and Spectrofluorimetric Determination of Netilmicin: A Sustainable Analytical Approach

Netilmicin (NTM) lacks an intrinsic chromophore, limiting its direct spectrophotometric determination. This study presents Eosin Y complexation as a simple, extraction-free strategy for the spectrophotometric and spectrofluorimetric determination of NTM in ophthalmic formulations and spiked artificial aqueous humor. The spectrophotometric method was linear over 0.40–25.00 μg mL −1 (r = 0.9999), with a detection limit of 0.132 μg mL −1 , while the spectrofluorimetric method was linear over 0.05–2.00 μg mL −1 (r = 0.9999), with a detection limit of 0.015 μg mL −1 . Both methods demonstrate satisfactory accuracy and precision according to ICH Q2(R2), with RSD values below 2% and mean recoveries of 101.95 ± 0.32 and 98.99 ± 0.99 for the spectrophotometric and spectrofluorimetric methods, respectively, in the commercial ophthalmic formulation. Recoveries in spiked artificial aqueous humor ranged from 99.65 ± 1.27 to 100.03 ± 1.32 for the spectrophotometric method and from 98.88 ± 0.65 to 101.13 ± 1.50 for the spectrofluorimetric method. The proposed methods require neither extraction nor organic solvents and provide simple and rapid procedures for routine NTM analysis. Their environmental and analytical characteristics were further assessed using complementary greenness, whiteness, blueness, ChlorTox, and RAPI metrics, providing multidimensional assessment of the environmental, practical, chemical-risk, and analytical-performance characteristics of the proposed methods.