| Cephalosporin drugs are beta-lactam antibiotics, with the characteristics of penicillinase-resistance, high efficacy, low toxicity, less allergic reactions and broad anti-bacterial spectrum, which can effectively inhibit bacterial growth and reproduction, treat a wide variety of bacterial infections in livestock and aquatic products. Trace amounts of antibiotics may remain in animal body due to improper use, which leads to an increasing potential risk for human health through the food chain. At present, strict limit requirements have been set by many countries, but only a few cephalosporins can be detected, which fails to meet the practical need of rapid detection for multi-cephalosporins. So it is necessary to establish an easy, quick, accurate and sensitive method for simultaneously detecting residues of multi-cephalosporins in animal products. In this paper, a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analytical method was developed for the simultaneous determination of12cephalosporins (Cefapirin, Cefquinome, Cefalonium, Cefotaxime, Cefalexin, Cefradine, Cefuroxime, Cefazolin, Cefoperazone, Cefalothin, Ceftriaxone, Cefacetrile), and a gel permeation chromatography (GPC) cleanup method for cephalosporins in animal products was also set up. Based on the established instrument method and the sample pretreatment method, a high sensitivity, high specificity and generic gel permeation chromatography-liquid chromatography tandem mass spectrometry method for determination of cephalosporins was established and applied to the animal products.1. Establishment of HPLC-MS/MS method for the simultaneous determination of12cephalosporins. As for the purpose of high sensitivity, the HPLC conditions and MS operation parameters were optimized. The response and peak shape of analytes were compared when methanol and acetonitrile was used as the LC mobile phase, and three different formic acid concentrations (0.05%,0.1%,0.2%, v/v) in aqueous phase were examined for the effect on the ionization efficiency of the analytes.0.1%(v/v) formic acid in water and methanol were used as mobile phases, and their gradient conditions were optimized. Using the positive electrospray ionization mode, the standard solution of single drug at5μg/mL was identified as the molecular ion by MS, which was further confirmed as fragment ions by MS/MS. To obtain maximum intensity for the molecular-characteristic ion pairs, many parameters, such as fragmentor voltage and collision energy, were also optimized. The linear relationship and precision of the method were then studied, the result showed that good linear correlations and precisions existed in the range of2.5-200ng/mL.2. Optimization of GPC cleanup. On Sephadex LH-20(320mm×16mm i. d) chromatographic column, tailed chromatographic peak and long retention time were found when cephalosporins were eiuted with methanol at a flow rate of2.0ml/min. Three acetonitrile concentrations (2.5%,5%,7.5%, v/v) in methanol were examined respectively, and5%(v/v) acetonitrile in methanol inhibited trailing and reduced the peak time effectively. Different ammonium acetate concentrations (0.5,2,3.5mmol/L) were then evaluated, and the optimal mobile phase was5%(v/v) acetonitrile in methanol, containing2mmol/L ammonium acetate. Using beef as materials, the separation efficiencies of the sample extracts and the cephalosporins standard solution on GPC were analyzed at three different flow rates of2.0,3.0,4.0mL/min. The result indicated that an optimal separation of cephalosporins and sample matrix was obtained at a flow rate of2.0ml/min, using methanol-acetonitrile (95:5, v/v,2mmol/L ammonium acetate) as the mobile phase.3. Study of sample matrix effects. The effects of sample matrices on detecting results were examined, and the matrix interference was diminished by preparing the standard solution with blank matrix. The post-extraction standard addition method was applied to investigate the matrix effects by the ratio of peak areas of blank matrix standard solution and methanol-water (3:7, v/v) standard solution. The sample matrix effects were removed by calibrating the sample addition solution with matrix standard solution, both of which have the same ions environment and competition. By this way, the true recoveries of cephalosporins were obtained, the accuracy and precision of quantitative analysis were assured.4. Development of GPC/HPLC-MS/MS. Beef was chosen as the research object, the extraction methods for cephalosporins in beef were optimized. Combining the best extraction method with the optimal GPC and HPLC-MS/MS, a suitable GPC/HPLC-MS/MS method for the determination of cephalosporins residue in beef was developed:the sample was extracted by acetonitrile-water (4:1, v/v), cleaned up by Sephadex LH-20gel column (320mm×16mm i. d), the separation of cephalosporins was performed on a CAPCELL PAK MG C18(100mm×2.0mm,3μm) column using a mobile phase of0.1%formic acid-methanol by gradient elution at a flow rate of0.2mL/min, the electrospray was operated under the positive ionization mode and all analytes were identified under selective reaction monitoring (SRM) mode, the calibration curves showed good linearities in the range of2.5～200ng/mL for the analytes with correlation coefficients of above0.9989, meeting the requirements of quantitative analysis, and the limits of detection(LOD, S/N>3) for cephalosporins in beef were0.5～1.5ng/g. Additional experiments showed the method of detecting residues of cephalosporin antibiotics in beef had a wide range of application, for example, it could be also used for detecting residues of cephalosporin antibiotics in pork, fish, chicken and loach. With a high sensitivity and a good generic, the method has a good application prospect. |
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