LAUSR

Abstract Tulathromycin is a macrolide antibiotic generally used for the treatment of respiratory diseases in cattle and swine. This work proposes an improvement of a previously published LC‐MS/MS method for tulathromycin determination in pig serum, here validated in three different bull matrices: plasma, seminal plasma, and urine. The approach is based on a quick protein precipitation with acetonitrile, filtration, and sample dilution before injection, allowing to rapidly process large batches of samples. Analytes separation was obtained using a BEH C18 (50 × 2.1 mm, 1.7 μm) column, maintained at 40°C with a chromatographic run of 5 min. The method was fully validated over concentration ranges suitable for field levels of tulathromycin found in each matrix (0.01–1 μg/ml for plasma, 0.05–5 μg/ml for seminal plasma, and 0.1–10 μg/ml for urine), showing good linearity during each day of testing (R 2 always >0.99). Accuracy and precision were within ±15% at all QC concentrations in all the three matrices. Furthermore, the use of tulathromycine‐d7 as internal standard mitigated the potential impacts of matrix effect. The validated technique was successfully applied to samples collected during a pharmacokinetic study in bulls, allowing to monitor tulathromycin concentrations over time in the three matrices. To our knowledge, this is the first validated approach for LC‐MS/MS quantification of tulathromycin in seminal plasma and urine.