Moretti Simone, Dusi Guglielmo, Giusepponi Danilo, Pellicciotti Simona, Rossi Rosanna, Saluti Giorgio, Cruciani Gabriele, Galarini Roberta
Istituto Zooprofilattico Sperimentale dell'Umbria e delle Marche, Via G. Salvemini, 1, 06126 Perugia, Italy.
Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna "B. Ubertini", Via A. Bianchi, 7/9, 25124 Brescia, Italy.
J Chromatogr A. 2016 Jan 15;1429:175-88. doi: 10.1016/j.chroma.2015.12.021. Epub 2015 Dec 11.
A multiclass method for screening and confirmatory analysis of antimicrobial residues in muscle has been developed and validated, according to Commission Decision 2002/657/EC. Sixty-two antibiotics belonging to ten different drug families (amphenicols, beta-lactams, diamino-pyrimidine, lincosamides, macrolides, pleuromutilins, quinolones, rifamycins, sulfonamides and tetracyclines) have been included in the method. After the addition of an aqueous solution of EDTA, the minced muscle was extracted with acetonitrile/water mixture and, later, with pure acetonitrile. The extract was evaporated and redissolved in ammonium acetate buffer prior to LC injection. Instrumental determination was performed by liquid chromatography coupled to hybrid high resolution mass analyser (LC-HRMS/MS) operating in positive electrospray ionization mode. Chromatographic separation was optimized on a Poroshell 120 EC-C18 column (100 × 3.0 mm, 2.7 μm) with gradient using methanol and water containing 0.1% of formic acid as mobile phases. The method was validated in bovine muscle in the range 3.3-150 μg kg(-1) for all antibiotics; for some compounds with MRL higher than 100 μg kg(-1), the validation interval has been extended until 1500 μg kg(-1). The studied performance characteristics were selectivity, linearity, precision, trueness (recovery), decision limits, detection capabilities, detection and quantification limits. Satisfactory quantitative performances were obtained for all the analytes. Ruggedness tests demonstrated the applicability to swine and poultry muscle, too. Finally the wide participation in proficiency tests allowed to investigate in deep the method performances.
根据委员会第2002/657/EC号决定，已开发并验证了一种用于肌肉中抗菌药物残留筛查和确证分析的多类别方法。该方法涵盖了属于十个不同药物家族的62种抗生素（氯霉素类、β-内酰胺类、二氨基嘧啶类、林可酰胺类、大环内酯类、截短侧耳素类、喹诺酮类、利福霉素类、磺胺类和四环素类）。加入乙二胺四乙酸水溶液后，将切碎的肌肉先用乙腈/水混合物萃取，随后用纯乙腈萃取。提取物经蒸发后，在进行液相色谱进样前重新溶解于醋酸铵缓冲液中。仪器测定采用液相色谱与混合高分辨率质谱仪联用（LC-HRMS/MS），以正电喷雾电离模式运行。在Poroshell 120 EC-C18柱（100×3.0 mm，2.7μm）上进行色谱分离优化，使用甲醇和含0.1%甲酸的水作为流动相进行梯度洗脱。该方法在牛肌肉中针对所有抗生素在3.3 - 150μg kg⁻¹范围内进行了验证；对于一些最大残留限量高于100μg kg⁻¹的化合物，验证区间已扩展至1500μg kg⁻¹。所研究的性能特征包括选择性、线性、精密度、准确性（回收率）、决策限、检测能力、检测限和定量限。所有分析物均获得了令人满意的定量性能。耐用性测试表明该方法也适用于猪和家禽肌肉。最后，通过广泛参与能力验证试验深入研究了该方法的性能。
