Imran Muhammad, Tawab Abdul, Rauf Waqar, Rahman Moazur, Khan Qaiser Mehmood, Asi Muhammad Rafique, Iqbal Mazhar
National Institute for Biotechnology & Genetic Engineering (NIBGE), PO Box 577, Jhang Road, Faisalabad, 38000, Pakistan.
Nuclear Institute for Agriculture and Biology, Jhang Road, Faisalabad, 38000, Pakistan.
J Chromatogr B Analyt Technol Biomed Life Sci. 2017 Sep 15;1063:163-173. doi: 10.1016/j.jchromb.2017.08.029. Epub 2017 Aug 24.
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 25-600μg/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 126μg/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 5days of oral dosing.
氟苯尼考是一种属于甲砜霉素类的广谱抑菌抗生素，广泛用于家禽和家畜的各种感染治疗。氟苯尼考在不同动物物种中的主要代谢产物是氟苯尼考胺，它被用作测定氟苯尼考的标记残留物。本研究表明，仅通过溶剂萃取分析氟苯尼考无法确定经处理禽类的目标组织（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号决定，开发并验证了一种基于液相色谱-串联质谱的可靠确证方法，用于分析鸡肉中氟苯尼考及其代谢产物。该方法基于酸水解，以释放可能与组织共价结合的不可萃取残留物，并将所有氟苯尼考残留物及其代谢产物转化为氟苯尼考胺。随后在pH 10.5条件下用乙酸乙酯回收胺，脱脂并通过分散固相萃取（dSPE）进一步净化。在反相C-18柱上使用乙腈/水流动相进行等度洗脱实现液相色谱分离，并在线性离子阱质谱仪上进行分析。在25-600μg/kg的浓度范围内获得了鸡肉的校准曲线。准确度值范围为84%至101.4%，日内和日间精密度值分别为1.2%-11.7%。检测限（LOD）、定量限（LOQ）、CCα和CCβ值分别为0.98、3.2、113和126μg/kg。所开发的方法具有高度稳健性，并进一步应用于估计口服给药5天后肉鸡肌肉、肝脏和肾脏样品中溶剂可萃取与溶剂不可萃取氟苯尼考药物残留的相对分布。
