Research Group of Bioanalysis, Department of Analytical Chemistry, University of Barcelona, Avda. Diagonal 645, E-08028 Barcelona, Spain; Research Group of Bioanalysis, Department of Analytical Chemistry, Food and Nutrition Torribera Campus, University of Barcelona, Avda. Prat de la Riba171, E-08921 Sta. Coloma de Gramenet, Barcelona, Spain.
Research Group of Analytical Chemistry and Life Sciences, Department of Analytical Chemistry, Campus of Fuentenueva, University of Granada, E-18071 Granada, Spain.
J Chromatogr A. 2014 Aug 22;1356:10-22. doi: 10.1016/j.chroma.2014.06.034. Epub 2014 Jun 20.
An analytical method based on a sample treatment by dispersive liquid-liquid microextraction (DLLME) followed by ultra high performance liquid chromatography-tandem mass spectrometry analysis (UHPLC-MS/MS) for the determination of 17 quinolones and 14 β-lactams (penicillins and cephalosporins) in raw cow milk, was validated according to the European Commission guidelines as cited in the Decision 2002/657/EC. The extraction efficiency of the DLLME depends on several parameters such as the nature and volumes of extractant and dispersive solvents, pH, concentration of salt, shaking time and time of centrifugation. These variables were accurately optimized using multivariate optimization strategies. A Plackett-Burman design to select the most influential parameters and a Doehlert design to obtain the optimum conditions have been applied. Two different pH values were used for the extraction of compounds (pH 3 for acidic quinolones and β-lactams and pH 8 for amphoteric quinolones). The method was validated using matrix-matched standard calibration followed by a recovery assay with spiked samples. The limits of quantification found ranged from 0.3 ng g(-1) for amoxicillin to 6.6 ng g(-1) for ciprofloxacin, and the precision was lower than 15% in all cases as is required by the European Regulation. The decision limits (CCα) ranged between 4.1 and 104.8 ng g(-1), while detection capabilities (CCβ) from 4.2 to 109.7 ng g(-1). These values were very close to the corresponding maximum residue limits (MLRs) for the studied antibiotics. Recoveries between 72 and 110% were also obtained. Finally, in order to evaluate the applicability of the method, 28 raw cow milk samples were analysed and it was observed that 28% of the samples were positive. However, only 11% were considered non-compliant with the current EU legislation (Commission Regulation 37/2010), due to some milk samples corresponded to treated cows with these antibiotics.
基于分散液相微萃取（DLLME）处理样品后，采用超高效液相色谱-串联质谱分析（UHPLC-MS/MS）的方法，对生牛乳中的 17 种喹诺酮类和 14 种β-内酰胺类（青霉素类和头孢菌素类）进行了测定。该方法按照欧洲委员会在委员会条例 2002/657/EC 中的指导原则进行了验证。DLLME 的萃取效率取决于多个参数，如萃取剂和分散溶剂的性质和体积、pH 值、盐浓度、振摇时间和离心时间。这些变量使用多元优化策略进行了精确优化。采用 Plackett-Burman 设计选择最有影响的参数，采用 Doehlert 设计获得最佳条件。对于化合物的提取，使用了两种不同的 pH 值（pH3 用于酸性喹诺酮类和β-内酰胺类，pH8 用于两性喹诺酮类）。该方法采用基质匹配标准校准，并用加标样品进行回收率测定进行验证。定量限范围为 0.3ng g(-1)（阿莫西林）至 6.6ng g(-1)（环丙沙星），所有情况下的精密度均低于 15%，符合欧洲法规的要求。决策限（CCα）范围为 4.1 至 104.8ng g(-1)，检测能力（CCβ）范围为 4.2 至 109.7ng g(-1)。这些值与研究抗生素的相应最大残留限量（MLRs）非常接近。回收率在 72%至 110%之间。最后，为了评估该方法的适用性，对 28 份生牛乳样品进行了分析，结果发现 28%的样品呈阳性。然而，只有 11%的样品被认为不符合当前欧盟法规（委员会条例 37/2010），因为一些牛奶样品对应于使用这些抗生素治疗的奶牛。
