Preparation Of Classs-specific Molecularly Imprinted Polymers For Macrolides And Its Application

A method based on liquid chromatography-tandem mass spectrometry?LCMS/MS?coupled with molecularly imprinted solid phase extraction was developed for the determination of ten macrolides drugs.Molecularly imprinted polymers?MIPs?were synthesized by bulk polymerization using tylosin,a 16-membered ring macrolide antibiotic,as the virtual template.And then the polymers were used to prepare molecularly imprinted solid phase extraction?MISPE?cartridge.Macrolides compounds in muscles samples were extracted with ethyl acetate,and then the MISPE cartridge was used for the separation,purification and enrichment of macrolides.After drying the eluent,20% methanol in 0.1% formic acid aqueous solution was used to re-dissolve the residues,and then target analytes were analyzed by LC-MS/MS.Acetonitrile and 0.1% formic acid were used as mobile phases,and macrolides drugs were separated using an Agilent Zorbax SB-Aq column?150 mm × 2.1 mm i.d.,3.5 ?m?with gradient elution program.Analysis was accomplished using multiple reaction monitoring?MRM?in positive electrospray ionization mode.Analytes were quantified using the matrix-matched external calibration curves.The experimental results showed that there were good correlative relationship for ten analytes in the linear ranges of 0.1²00 ?g kg^-1?r2? 0.99?.The limits of detection?LODs?were in the ranges of 0.1⁰.4 ?g kg^-1 and the limits of quantification?LOQs?were 0.3¹.0 ?g kg^-1.In the three spiked concentrations of 1,5 and 20 ?g kg^-1,recoveries of ten macrolides in swine,cattle and chicken muscles ranged from 60.7% to 100.3%.The intra-day relative standard deviations were 0.2%¹4% and inter-day were 2.4%¹4%.The results also indicated that the MISPE cartridge has the characteristic of class-specific.Compared with C18,SCX and Oasis HLB cartridges,the MISPE cartridge exhibited better purification and concentration effects.The class-specific molecularly imprinted polymers were synthesized by precipitation polymerization using tulathromycin,a 15-membered ring macrolide antibiotic,as the virtual template and methacrylic acid as the functional monomer.Through optimization the system of polymerization,the optimal formula was as follows: 0.1 mmol tulathromycin?template?,0.4 mmol methacrylic acid?functional monomer?,2 mmol ethylene glycol dimethacrylate?cross-linker?,8 m L chloroform and 12 m L acetonitrile?porogens?and 10 mg azobisisobutylnitrile?initiator?.The physical and chemical properties of the polymers were characterized by scanning electron microscopy,infrared spectroscopy,specific surface and thermal gravimetric analysis.The static adsorption test showed that the MIPs had more large adsorption capacity than the non-imprinted polymers?NIPs?,and the maximum adsorption capacity was 50.8 mg g-1.Dynamic adsorption test showed that the MIPs reached adsorption equilibrium after 4 h,and had longer adsorption equilibrium time and greater adsorption capacity than the NIPs.The class-specific adsorption test suggested that the specificity of the polymers was not only related to the ring structure of macrolides,but also with the link location of side chain groups.Using the polymers prepared as the dispersed solid phase extraction adsorbent,a method for the simultaneous determination of ten macrolides in pork by LC-MS/MS coupled with class-specific molecularly imprinted dispersive solid phase extraction was established.Macrolides residues in pork sample were extracted with 1% acetic acid in acetonitrile,then adsorbed with the MIPs,and finally the analytes absorbed in the polymer particles were desorbed with 10% acetic acid in methanol.The results showed high correlation coefficients of ten analytes in the linear ranges of 0.1²00 ?g kg^-1 and the correlation coefficients?r2?were higher than 0.99.The LODs and LOQs were in the ranges of 0.1¹.0 ?g kg^-1 and 0.3⁵.0 ?g kg^-1,respectively.In the three spiked concentrations of 5,10 and 25 ?g kg^-1,the recoveries of ten analytes in MIPs were 51.8%⁹2.8%,and NIPs were 41.6%⁵6.9%.The intra-day relative standard deviations were 1.0%¹2% and inter-day were 2.8%¹0%.