Liu Rong, Tang Xiaoling, Xiong Renping, Li Lu, Du Xiaoxi, He Limin
Quality Supervision, Inspection and Testing Center for Domestic Animal Products (Guangzhou), Ministry of Agriculture and Rural Affairs, Guangzhou 510642, China.
National Reference Laboratory of Veterinary Drug Residues (SCAU), College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Mar 15;1193:123169. doi: 10.1016/j.jchromb.2022.123169. Epub 2022 Feb 12.
Illegal drug residues in animal derived foods are closely related to human's life and health. Studies on illegal drug residues and the metabolism, such as β-agonists in animals have attracted more and more attention. In most cases, β-agonists are suppliedand used astheracemate. The metabolic process and distribution of the two enantiomers in animal tissues are different. Therefore, it is very necessary to develop a simple and fast method for chiral resolution of these drugs in animal tissues. In this paper, a reliable resolution and determination method was presented using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for fourteen enantiomers of seven β-agonist racemates, clenbuterol (CLE), salbutamol (SAL), cimaterol (CIM), terbutaline (TER), clorprenaline (CLO), tulobuterol (TUL), penbuterol (PEN) in pork, beef, and lamb muscle samples. The samples were added the internal standard solution (IS) and extracted in the alkaline medium with acetonitrile. The further sample purification was accomplished through MCX solid phase extraction cartridge. Chromatographic chiral separation was carried out on a VancoShell chiral column (100 mm × 4.6 mm, 2.7 μm) with an isocratic mobile phase consisting of methanol and 10 mmol mL ammonium formate aqueous solution (85:15, v/v). Under the optimized conditions, the resolution (R) of CIM was 2.0, CLE and PEN were 1.5, the others were all greater than 1.0. Enantiomeric determination was performed in the positive electrospray ionization mode using multiple reaction monitoring (MRM). The correlation coefficient (r) in the range of 0.2-25.0 μg L was above 0.993. The average recoveries at the three spiking levels ranged from 95.3% to 117.7% with the relative standard deviation (RSD) lower than 15%. The limit of detection (LOD) and the limit of quantification (LOQ) of β-agonist enantiomers was 0.2 μg kg and 0.5 μg kg respectively. The method was successfully applied in the analysis and evaluation of β-agonist enantiomers in positive food animal muscle samples, CLE, SAL, TEB and CIM enantiomers were detected. The concentrations of the corresponding enantiomers were in the range of 1.06-17.3 μg kg, the lowest enantiomer fraction (EF) value was 0.42, and the highest value was 0.69. The work is expected to provide a method for chiral separation and enantiomeric determination of the further study of pharmacology, toxicity and residue elimination of β-agonist enantiomers.
动物源性食品中的非法药物残留与人类生命健康密切相关。对动物体内非法药物残留及其代谢情况（如β-激动剂）的研究越来越受到关注。在大多数情况下，β-激动剂是以消旋体形式供应和使用的。两种对映体在动物组织中的代谢过程和分布不同。因此，开发一种简单快速的方法用于动物组织中这些药物的手性拆分非常必要。本文提出了一种可靠的拆分和测定方法，采用液相色谱-串联质谱法（LC-MS/MS）测定猪肉、牛肉和羊肉肌肉样品中7种β-激动剂消旋体（克伦特罗（CLE）、沙丁胺醇（SAL）、西马特罗（CIM）、特布他林（TER）、氯丙那林（CLO）、妥布特罗（TUL）、喷布特罗（PEN））的14种对映体。样品加入内标溶液（IS）后，在碱性介质中用乙腈萃取。通过MCX固相萃取柱进一步进行样品净化。在VancoShell手性柱（100 mm×4.6 mm，2.7μm）上进行色谱手性分离，等度流动相由甲醇和10 mmol/L甲酸铵水溶液（85:15，v/v）组成。在优化条件下，CIM的分离度（R）为2.0，CLE和PEN为1.5，其他均大于1.0。采用正电喷雾电离模式，通过多反应监测（MRM）进行对映体测定。在0.2 - 25.0μg/L范围内相关系数（r）大于0.993。三个加标水平的平均回收率为95.3%至117.7%，相对标准偏差（RSD）低于15%。β-激动剂对映体的检测限（LOD）和定量限（LOQ）分别为0.2μg/kg和0.5μg/kg。该方法成功应用于阳性食用动物肌肉样品中β-激动剂对映体的分析和评估，检测到了CLE、SAL、TEB和CIM对映体。相应对映体的浓度范围为1.06 - 17.3μg/kg，最低对映体分数（EF）值为0.42，最高值为0.69。该工作有望为β-激动剂对映体的药理学、毒理学及残留消除的进一步研究提供一种手性分离和对映体测定方法。
