Abstract
IMPORTANCE: Gentamicin is an aminoglycoside antibiotic used to treat infections in dairy cows. Because of its potential toxicity and milk residue risk, an evidence-based withdrawal period is essential to ensure consumer safety. OBJECTIVE: This study aimed to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify gentamicin residues in milk and to establish a withdrawal period for an intramuscular gentamicin product in dairy cows. METHODS: Nine Holstein cows received a gentamicin product (50 mg gentamicin sulfate/mL) at 0.1 mL/kg twice daily at 12 h intervals via intramuscular injection. Milk samples were collected before dosing and at 12 h intervals up to 168 h after the final administration and analysed with a validated LC-MS/MS method using heptafluorobutyric acid as an ion-pairing reagent. RESULTS: The method showed recovery rates of 86%-104%, coefficients of variation of 3%-11%, and limits of detection and quantitation of 23 and 70 ng/mL, respectively. Mean gentamicin concentrations exceeded the maximum residue limit (MRL; 0.2 mg/kg) at 12 h during treatment and 12 h after the final administration, and were < MRL in all cows from 36 h after the last dose. Based on residue depletion data and a national non-statistical guideline, a withdrawal period of 40-48 h was derived, and the regulatory withdrawal period for this product was established as 3 days. CONCLUSIONS AND RELEVANCE: The validated LC-MS/MS method enables rapid quantitation of gentamicin residues in bovine milk. A 3-day regulatory withdrawal period for this product supports milk safety, regulatory compliance, and practical use in dairy herds.