Determination of Gentamicin: Development and Validation of a Sensitive UPLC-MS/MS Assay According to the European Medicines Agency Guideline

Background/Objectives: Gentamicin (GEN) is an aminoglycoside antibiotic used in veterinary medicine to treat infections caused mainly by Gram-negative bacteria. GEN is a mixture of pharmacologically active components, known as isoforms. The objective was to develop and validate a sensitive, accurate, and precise Ultra-Performance Liquid Chromatography with triple quadrupole mass detector (UPLC-MS/MS) method to quantify the different GEN isoforms in pig plasma and feces using streptomycin as an internal standard. Methods: Solid-phase extraction (SPE) was carried out. A high-strength silica (50 × 2.1 mm, 1.8 µm) column was used for chromatographic separation and a mobile phase of 0.26% HFBA in water (A) and acetonitrile (B) was delivered in a gradient with a flow rate of 0.5 mL/min. The column temperature was 40 °C and the sample injection volume was 30 µL. Results: The method showed good selectivity and specificity, with no interfering peaks. Calibration curves were linear in the range from 0.05 to 0.3 µg/mL for all isoforms in both matrices. Within- and between-run precision and accuracy were satisfactory for the lower limit of quantification (LLOQ), with coefficients of variation (CV) ≤ 13.4% and deviations ≤ 116.5% in plasma and CV ≤ 12.3% with deviations ≤ 101.7% in feces. No carry-over was observed, and analyte stability was confirmed under different storage conditions. Conclusions: The method development fulfilled all validation criteria established by the European Medicine Agency Guideline (EMA/CHMP/ICH/172948/2019). Moreover, the applicability of the method in clinical practice was demonstrated by the quantification of GEN in plasma and feces samples from pigs.