Analysis Of Macrolide Antibiotic Residues By Capillary Electrophoresis And Molecularly Imprinted Solid Phase Extraction

Macrolide antibiotics(MALs)is one of the widely used antibiotics,not only as a therapeutic drug in human clinical medicine and livestock breeding,but also as an arisen feed additive.However,unreasonable or excessive abuse will result in environmental and animal food residues,and finally endanger the health of human being through the food chain.Therefore,it is necessary to establish a high sensitive detection method to monitor the MALs residue in food.This paper chose five kinds of MALs including tilmicin,erythromycin,roxithromycin,clarithromycin and tylosin as analytes,and established a new detection method and sample pretreatment method.The related research contents are detailed as following:(1)A new capillary electrophoresis method was developed for the detection of five kinds of MALs residues in milk by micellar electrokinetic capillary chromatography with field amplified sample stacking.The main factors that affect the peak area,resolution and enrichment factors were optimized.The optimized running buffer contained 60 mM PBS,5 mM sodium cholate,and 1.0 mM CTAB with pH 7.0.The separation voltage was 20 kV,column temperature was 25 ℃ and the sample was injected at 10 k V for 120 s.The detection wavelength was 195 nm.Under the optimized conditions,the enrichment factor of tilmicosin,erythromycin,clarithromycin,roxithromycin and tylosin were 866,565,908,702 and 675,respectively.The limits of detection(LODs,S/N = 3)ranged from 0.002-0.004 mg/L.The average recovery was between 72.8%-93.7%.The new method meet the requirement of maximum residue limits of these five antibiotics of China,the European Union,the United States and so on,and can be applied to the detection of the macrolide antibiotics residues in milk.(2)A method for the preparation of molecularly imprinted polymers(MIP)by precipitation polymerization was established,using erythromycin and tylosin as template,methacrylic acid(MAA)as functional monomer,ethylene glycol two methyl acrylate(EGDMA)as crosslinking agent.The proportion of the two templates,functional monomer and crosslinking agent,the type and the amount of porogen were optimized,and the adsorption performance of the synthesized MIP was characterized.The optimal preparation conditions are as follows: n(ERY/TYL): n(MAA): n(EGDMA)= 1 : 6 : 10(where n(ERY): n(TYL)= 1 : 1),methanol / acetonitrile(1:3,V/V,2 mL)as solvent,azobisisobutyronitrile(AIBN,0.02 g)as initiator,polymerize at 60 ℃ for 24 h.The dissociation constants of high and low affinity’s binding sites are 1.379 and 4.630 mmol/L for ERY,3.650 and 17.857 mmol/L for TYL,where the max apparent binding capacities are 135.466 and 291.907 μmol/g for ERY,138.704 and 523.054 μmol/g for TYL.The index of imprinting-induced promotion of binding is 93.534% and 125.856% for TYL and ERY,respectively.More importantly,these good adsorption performances can be remained even recycling use for 10 times.(3)A method was developed for the pretreatment of milk samples based on molecularly imprinted solid phase extraction(MISPE)column using above mentioned MIP as filler.The optimal preparation conditions are as follows: the loading solution was 20% acetonitrile in water(5 m L),the washing solution was 10% methanol in water(4 m L),and the eluent was 5% ammonia in methanol(5 m L).The recoveries of 5 kinds of MALs were 75.3%-94.2%,higher than HLB column,and the enrichment ratio combined with field amplified sample stacking micellar electrokinetic capillary chromatography can reach 2825 to 4540,with LOD 0.3 to 0.8 μg/L.Therefore,the MISPE column can be used in the enrichment and purification of milk samples.