Determination Of Penicillins In Milk By6-APA MISPE-HPLC

Penicillin antibiotics are now widely used in the prevention and treatment of mastitis in dairycow. Improper use or violation of withdrawal period will cause antibiotic residues, which will affectthe quality of milk and harm human health as well. Antibiotic residues have also become theinternational trade green barrier. International society is paying more attention to the pretreatment anddetection technology, as well as detection and extraction research of animal derived food residues.The aim of this study was to use penicillin intermediate6-amino penicillanic acid (6-APA) asvirtual template to prepare penicillins (PENs) molecular imprinted solid phase extraction (MISPE)column and to extract multiple types of penicillin from complex matrix of biological samples,including penicillin potassium (penicillin G, PEN), ampicillin (AMP), amoxicillin (AMO) andintermediate6-APA. High performance liquid chromatography (HPLC) was used for simultaneousseparation and detection. The established MISPE-HPLC method provides a new pretreatment anddetermination technique for detection of trace analytes in complex matrix sample. Another ultrahighperformance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) technique wasestablished to simultaneously detect four PENs, which could be foundation for further study.The research contents are as follows:1. Preparation of6-APA molecular printed polymer and MISPE columnThe6-APA molecular printed polymer was prepared using parent nucleus of penicillin6-APA astemplate molecule, α-methacrylic acid (MAA) as functional monomer, ethylene glycol dimethacrylate(EDMA) as cross-linking agent, and formic acid as porogen (polymerization ratio1:5:30:0.3) byprecipitation polymerization. The structure and morphology of this polymer were characterized usinginfrared spectroscopy and scanning electron microscopy. The adsorption property of the polymer wasstudied by static adsorption experiment. Scachard analysis showed that this polymer has larger surfaceadsorption capacity on three types of template molecule analogues: AMO, PEN and AMP.The prepared molecular imprinted polymer was mixed with diatomite in a ratio of2:1in theempty SPE column to prepare MISPE. The optimal activating and eluting agents were determinedafter comparison of effect on column by different agents. The recovery rate was between83.5-87.27%.2. Establishment of HPLC methodBy comparing separation effects of different types and ratio of mobile phase, the HPLC methodwas established to simultaneously detect6-APA, AMP, AMO and PEN using Xterra MS C18column,acetonitrile:0.1%ammonia (5:95) as mobile phase and wavelength of200nm. Separation can beachieved in6min. The method has a good linear relationship in0.125-10mg/L with a correlationcoefficient r2of0.9972-0.9993and a relative standard deviation (RSD) of2.01-2.22%(n=6).3. Establishment of high performance capillary electrophoresis (HPCE) methodHPEC method was established to simultaneously detect four PENs in milk. Experimentconditions were optimized using orthogonal experimental design with40mmol/L potassiumdihydrogen phosphate-20mmol/L borax buffer solution (pH7.8), separation voltage of28kV,separation temperature of30℃and time of4.5min. It has a good linear relationship between1.56-100mg/L with a correlation coefficient r2of0.9979-0.9998and a relative standard deviation(RSD) of1.20-1.93%(n=6), which can be used in detection in preparation of molecular imprintedpolymers. 4. Establishment of6-APA MISPE-HPLC methodThe effects on6-APA and template analogues recovery rates by types of MISPE columnactivation solvent, types and amount of eluting agent were studied on the prepared6-APA MISPEcolumn. The10ml3.2g/L bromide butyl ammonium acetonitrile was used to extract,3ml80%methanol solution was used as activation solvent and3ml acetonitrile-methanol (v:v,3:7) was used aseluting agent for purification in pretreatment method. The recovery rate was between83.5-87.27%.Combination of6-APA MISPE with HPLC can detect PENs in milk.Studies showed that this method was simple, quick, easy, highly selective, precise and accuratethat it can simultaneously detect4PENs within4min. The method has exploratory and practicalsignificance for the establishment of new detection method of residues in veterinary medicine andanimal derived foods.5. Establishment of UPLC-MS-MS methodBy comparing the separation effect of4PENs by different ratio of mobile phase, the optimalUPLC condition was determined as AgilentC18(2.1×150mm,5μm) column and mobile phase of75%water-25%acetonitrile.Mass spectrometry condition was set up as follows: electrospray ionization (ESI), negative ionscanning ionization mode,4000V capillary voltage,35psi atomizing air pressure,9.5L/min dry gasflow,350℃drying air temperature, MRM mode mass spectrometry, and high purity nitrogencollision gas. The capillary outlet voltage and collision energy was determined by molecularstructures of4PENs. Parent ion of each PENs was first determined by optimization in transmissionvoltage, then characteristic ion of each parent ion was determined by optimization in collision voltageto determine the quantitative ion. The method has a good linear relationship between0.15-5mg/L witha correlation coefficient r2of0.9992-0.9995. The limit of detection (LOD) and limit of quanlification(LOQ) of4PENs were0.02mg/L and0.05mg/L, respectively.