Enzyme immunoassay and liquid chromatography-fluorescence detection for amikacin in raw milk

An enzyme immunoassay and a liquid chromatography (LC) method for amikacin (AMK) in raw milk were developed in this study. Anti-AMK antibody was prepared by immunizing rabbits with AMK–BSA conjugate. The developed immunoassay exhibited an IC50 value of 1.30 ng/mL and the spiked recoveries at 25–1000 ng/mL ranged from 69.8% to 93.3% with coefficients of variation (CVs) of 8.5–17.6%. For LC analysis, AMK was derivatized with 9-fluorenylmethyl chloroformate, which was followed by C8 column separation and fluorescence detection. By trichloroacetic acid extraction and MCX column purification, the recoveries at spiked concentrations of 500–5000 ng/mL were 80.7–91.3% with CVs less than 6.3%. The two methods can be selectively used for rapidly screening or quantitatively determining AMK in raw milk.

► A polyclonal antibody against amikacin was prepared. ► An enzyme-linked immnunosorbent immunoassay for amikacin in milk was developed. ► A liquid chromatography method for amikacin in milk was developed. ► The two methods can complement each other for determining amikacin in milk.