Simultaneous detection and comparative pharmacokinetics of amoxicillin, clavulanic acid and prednisolone in cows’ milk by UPLC–MS/MS

• This is the first report of simultaneous determination of amoxicillin, clavulanic acid and prednisolone in cows’ milk using UPLC–MS/MS.
• The validated method has been successfully applied to a comparative pharmacokinetic study in healthy and mastitic cows.
• The extended elimination times of amoxicillin and prednisolone after intramammary infusion in mastitic cows were firstly reported.

Amoxicillin (AMOX), clavulanic acid (CLAV) and prednisolone (PSL) are widely used in combination for the treatment of mastitis in lactating dairy cows. However, no method has been reported to detect these three chemicals in milk in a single assay. In the present work, a reliable and sensitive UPLC–MS/MS method was developed and validated for simultaneous determination of AMOX, CLAV and PSL in cow’s milk. The analytes were determined by a positive and negative ionization electrospray mass spectrometer via multiple reaction monitoring. The linear ranges of AMOX, CLAV and PSL were from 2 to 1000 ng/mL, 20–1000 ng/mL and 1–1000 ng/mL, respectively, with the correlation coefficients greater than 0.999. The limits of quantification (LOQs) were 2 ng/mL (AMOX), 20 ng/mL (CLAV) and 1 ng/mL (PSL). Recoveries of the analytes of interest in milk samples were in the ranges of 84.2–101.4%. The intra-day and inter-day precisions ranged from 1.8% to 11.9%. This method was successfully applied to investigate the pharmacokinetics of AMOX, CLAV and PSL in milk from healthy and mastitic cows. The elimination times of AMOX and PSL in mastitic cows were longer than that in healthy cows, but the elimination times of CLAV did not show significant difference.