Florfenicol, a broad spectrum bacteriostatic antibiotic belonging to amphenicol class, is widely used in poultry and livestock for the treatment of various infections. The major metabolite of florfenicol in different animal species is florfenicol amine which is exploited as the marker residue for the determination of florfenicol. Analysis of florfenicol merely by solvent extraction cannot determine the accurate amount of the drug present in incurred tissues (muscle, liver and kidney) of treated birds, as indicated by this study. Thus the methods solely based on solvent extraction may lead to false negative results. A reliable LC–MS/MS based confirmatory method for the analysis of florfenicol and its metabolites in chicken muscle was developed and validated according to the European Union Commission Decision 2002/657/EC. The method was based on acid hydrolysis to liberate non-extractable residues having presumably been covalently bound to tissues, and to convert all the florfenicol residues as well as its metabolites into florfenicol amine. The amine was subsequently recovered with ethyl acetate at pH 10.5, defatted and further cleaned up with dispersive solid phase extraction (dSPE). The LC separation was achieved on reverse phase C-18 column with isocratic elution using acetonitrile/water mobile phase and the analysis was performed on linear ion trap mass spectrometer. Calibration curve was obtained over a concentration range of 25–600 μg/kg for chicken muscles. The accuracy values ranged from 84 to 101.4% and the precision values for within day and between days ranged from 1.2-11.7%, respectively. Limit of detection (LOD), limit of quantification (LOD), CCα and CCβ values were 0.98, 3.2, 113 and 126 μg/kg, respectively. The developed method was highly robust and was further applied to estimate the relative distribution of solvent-extractable against solvent-non-extractable florfenicol drug residues in muscle, liver and kidney samples of broiler chicken after 5 days of oral dosing.

氟苯尼考是属于安非尼类的广谱抑菌抗生素，广泛用于禽类和家畜中，用于治疗各种感染。氟苯尼考在不同动物物种中的主要代谢物是氟苯尼考胺，该胺被用作测定氟苯尼考的​​标记残留物。这项研究表明，仅通过溶剂萃取对氟苯尼考进行分析无法确定在治疗过的禽类的发生组织（肌肉，肝脏和肾脏）中药物的准确含量。因此，仅基于溶剂萃取的方法可能会导致假阴性结果。根据欧盟委员会第2002/657 / EC号决议，开发并验证了一种可靠的基于LC-MS / MS的验证方法，用于分析鸡肉中氟苯尼考及其代谢物。该方法基于酸水解，以释放可能与组织共价结合的不可提取的残基，并将所有氟苯尼考残留物及其代谢物转化为氟苯尼考胺。随后用pH 10.5的乙酸乙酯回收胺，脱脂，然后用分散固相萃取（dSPE）进一步净化。使用乙腈/水流动相在等度洗脱下在反相C-18柱上完成LC分离，并在线性离子阱质谱仪上进行分析。鸡肉的浓度在25–600μg/ kg的范围内获得了校准曲线。一天之内和两天之间的精度值范围从84％到101.4％，而精度值范围从1.2-11.7％不等。检测限（LOD），定量限（LOD），CCα和CCβ值分别为0.98、3.2、113和126μg/ kg。所开发的方法具有很强的鲁棒性，可进一步用于评估口服给药5天后肉鸡肌肉，肝脏和肾脏样品中可提取溶剂相对于不可提取氟苯尼考残留的相对分布。