Research On Determination Of Cefotaxime In Milk By MISPE-HPCE

As a kind of high-efficiency and wide-spectrum antibiotic, Cephalosporins are widely used in veterinary medicine for the treatment and prevention of diseases. Some improper use of drugs leads to residues in edible tissues which can cause health problems for individuals hypersensitive to them. The effective monitor becomes one of the most important procedures to ensure food safety. The research of Cephalosporins residues focuses on the methods of efficient , selective , simple and environment-friendly determination of the residues. The test selects CTX and CFR as factors and the following things are done:1. Optimizing the Method of Simultaneous Determination of CTX and CFR by HPCEThe test studies the effect of buffer concentration,buffer pH,running voltage and operating temperature to separation. The result of the orthogonal experiment analysis shows that the best condition is as follows:40mmol/L borax buffer (using 6mol/L hydrochloric acid adjusted to pH7.5),detection wavelength 210nm,running voltage 20 kV and operated temperature 20℃. Under such a condition,Cefotaxime(CTX)and Cefadroxil(CFR)can be separated completely in 12min. The liner range is 5μg/mL ~100μg/mL and the coefficients are 0.9994 to 0.9998. The RSDs of area are both lower than 4%.2. Preparation and Evaluation of MIPs of CTXA kind of molecular imprinted polymer is prepared by using Cefotaxime as the template molecule,MAA as functional monomer and EDMA as the cross linkers. The test studies effects of preparing MIPs including template molecule,functional monomer and cross linkers,etc. The best preparating condition of MIPs is fixed by the orthogonal experimen. The MIP is evaluated by binding experiment and Scatchard analysis.The results show that the best preparation codition is as follows:0.2mmol CTX,1.2mmol MAA,30mmol EDMA,4mL methanol/ acetonitrile(1/1) and 50℃and the MIP recognizes the template with two classes of binding sites. The data obtained in equilibrium adsorption experiments is processed by Scatchard analysis,the dissociation contents(KD) and the apparent maximum binding capacity (Qmax) are KD1= 0.367mmol/L ,Qmax1=6.06μmol/g,for low affinity binding sites and KD2=6mmol/L,Qmax2=36μmol/g,for high affinity binding sites,respectively. Selective absorption experiment results show that MIPs have higher affinity to CTX,compared with blank polymer and display a better selectivity. They also show high absorbability for CFR with similar structures.3. Filling the MISPE column to CTX and evaluation of the method of MISPEThe MISPE column is filled with MIPs and blank SPE column and the eluent and sample solvent are selected and optimized. The method of MISPE concentration and purification with CTX is established .The recovery rate is up to 79%～86%.4. Application of determination of CTX in milk by MISPE-HPCEThe result of the recovery rate for adding standard shows that the method of MISPE-HPCE is up to the requirement of determination of animal drugs residues. The recovery rate is in the range of 79%～85%.