Abstract: An enzyme inhibition method was established and used for multi-residue detection of β-lactamase inhibitors in milk. For sample preparation, the protein was precipitated with acetonitrile and the fat was removed with n-hexane. The matrix-matched external standard method was used for quantification, which eliminated the interference of the milk matrix. The detection limits of sulbactam, potassium clavulanate, tazobactam and avibactam sodium in milk samples were 42.88, 10.20, 1.68 and 3.48 μg/kg, respectively. The average spiked recoveries were from 80.43% to 115.87% with variation coefficients below 10.14%. When the residue of β-lactamase in milk did not exceed 40 U/mL, the assay results obtained using the developed method were accurate. Good agreement between the results of this method and those of liquid chromatography-tandem mass spectrometry (LC-MS/MS) was obtained for spiked milk samples, with a correlation coefficient (R2) of 0.982. The enzyme inhibition method allowed accurate, rapid, high-throughput and multi-residual detection of β-lactamase inhibitors, and was particularly suitable for on-site screening for β-lactamase inhibitor residues in large numbers of milk samples.

Key words: β-lactamase inhibitors; sulbactam; multi-residue detection; enzyme inhibition method; nitrocefin