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This study aimed to prepare a molecularly imprinted monolithic extraction column (MIMC) inside a micropipette tip by situ polymerization with roxithromycin as the dummy template. The polymers possessed excellent adsorption capacity and class-specificity to multiple macrolide drugs. MIMC was directly connected to a syringe for template removal and for the optimization of extraction conditions without any other post-treatment of polymers. A liquid chromatography-tandem mass spectrometric method was developed for the selective microextraction and determination of macrolide antibiotics in animal muscles based on MIMC. High recoveries of 76.1–92.8% for six macrolides were obtained with relative standard deviations less than 10.4%. MIMC exhibited better retention ability and durability when compared with the traditional C18 and HLB cartridges. The proposed method shows a great potential for the analysis of macrolide drugs at the trace level in animal foodstuffs.

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