Development of a chromatographic method for the separation and quantitative analysis of aminoglycoside group antibiotics

This study presents the development and validation of a high-performance liquid chromatography method coupled with charged aerosol detection (UHPLC-CAD) for the analytical characterization of veterinary medicinal products containing the aminoglycoside group antibiotics gentamicin, neomycin, and kanamycin. Due to their low UV absorbance and highly polar, ionic nature, aminoglycosides pose significant analytical challenges for conventional detection systems. The UHPLC-CAD method developed in this study effectively overcomes these limitations through enhanced selectivity, reproducibility, and operational simplicity. Each aminoglycoside was analyzed using a separately optimized protocol, with tailored column types, mobile phase compositions, flow rates, and detector settings. Method validation was conducted in accordance with ICH Q2(R1) guidelines, demonstrating excellent performance in terms of selectivity, linearity (R² > 0.995), accuracy, and precision (RSD < 2%). For instance, the limit of quantification (LOQ) for the gentamicin impurity sisomicin was determined as 1.9 µg/mL. In several commercial products, impurity levels such as sisomicin and neomycin C were found to exceed the limits defined by the European Pharmacopoeia. High Analytical Eco-Scale scores (n>75) confirmed that the developed methods in the study not only deliver technical robustness but also align with principles of environmental sustainability. The CAD-based UHPLC system eliminates the need for derivatization, minimizes matrix effects, and offers rapid analysis which are significant advantages for compounds with complex structures like aminoglycosides. In conclusion, this study goes beyond analytical method development, offering a regulatory-compliant, eco-conscious, and analytically sound strategy for pharmaceutical quality control. The findings provide a valuable foundation for revising current pharmacopeial standards and advancing future frameworks for the quality assessment of aminoglycoside antibiotics.