研制一种免疫亲和色谱柱，用于从饲料、肉类和肝脏样品中选择性提取新型激动剂苯乙胺 A。
Development of an immunoaffinity chromatography column for selective extraction of a new agonist phenylethylamine A from feed, meat and liver samples.
机构信息
The Key Lab of Health Chemistry & Molecular Diagnosis of Suzhou, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Renai Road 199, Suzhou 215123, China.
College of Pharmacy Sciences, Soochow University, Renai Road 199, Suzhou 215123, China.
出版信息
J Chromatogr B Analyt Technol Biomed Life Sci. 2014 Jan 15;945-946:178-84. doi: 10.1016/j.jchromb.2013.11.057. Epub 2013 Dec 3.
Phenylethanolamine A (PA) is a new emerged β-adrenergic agonist that has been illegally used as an animal feed additive for growth promotion in China. In this study, an immunoaffinity chromatography (IAC) column for selective extraction of PA from swine feed, meat and liver samples was developed. The IAC column was constructed by covalently coupling specific polyclonal antibody (Ab) against PA to CNBr-activated Sepharose 4B and packed into a common solid phase extraction (SPE) cartridge. The extraction conditions including loading, washing and eluting solutions were carefully optimized. Under optimal conditions, the IAC column was characterized in terms of maximum capacity, selectivity, extraction recovery and stability. The maximum capacity of the ICA for PA extraction was found to be 239.4ng. For selectivity testing, 100ng of other three β-adrenergic agonists (clenbuterol, ractopamine and salbutamol) was separately loaded onto the column, and it was observed that the tested compounds could not be captured on the column, e.g. the column could only selectively recognize PA. The recovery of the IAC for PA extraction was found within 96.47-101.98% when 10, 50 and 100ng PA were separately loaded onto IAC column. The IAC column was also applied to real sample extraction. Swine feed, meat and liver samples were collected and spiked with PA in range of 1.0-20ngg(-1). The spiked and unspiked samples were extracted by IAC column and measured by high performance liquid chromatography (HPLC). It was found that there was no detectable PA in the blank samples, and the extraction recoveries of the IAC for PA from the spiked samples were within 89.48-104.89%. The stability of the column was also tested. It was showed that after 35 times repeated usage, 60% of the maximum capacity was still remained. The proposed IAC was proven to be a feasible extraction method for PA from different matrices with the properties of high maximum capacity, selectivity, extraction efficiency and stability.
苯乙醇胺 A（PA）是一种新出现的β-肾上腺素激动剂，在中国已被非法用作动物饲料添加剂以促进生长。本研究开发了一种免疫亲和色谱（IAC）柱，用于从猪饲料、肉和肝样中选择性提取 PA。IAC 柱通过将针对 PA 的特异性多克隆抗体（Ab）共价偶联到 CNBr 活化的 Sepharose 4B 上构建，并填充到常见的固相萃取（SPE）小柱中。仔细优化了包括加载、洗涤和洗脱溶液在内的提取条件。在最佳条件下，从最大容量、选择性、提取回收率和稳定性方面对 IAC 柱进行了表征。IAC 对 PA 提取的最大容量为 239.4ng。为了进行选择性测试，将 100ng 的其他三种β-肾上腺素激动剂（克伦特罗、莱克多巴胺和沙丁胺醇）分别加载到柱上，结果表明这些测试化合物不能被柱捕获，即该柱只能选择性地识别 PA。当分别将 10、50 和 100ng 的 PA 加载到 IAC 柱上时，IAC 对 PA 提取的回收率在 96.47-101.98%范围内。该 IAC 柱还应用于实际样品提取。收集猪饲料、肉和肝样，并在 1.0-20ng/g 范围内添加 PA。用 IAC 柱提取加标和未加标样品，并用高效液相色谱法（HPLC）进行测量。结果表明，空白样品中未检测到 PA，IAC 从加标样品中提取 PA 的回收率在 89.48-104.89%范围内。还测试了柱的稳定性。结果表明，经过 35 次重复使用后，仍保留了 60%的最大容量。该研究表明，所提出的 IAC 是一种从不同基质中提取 PA 的可行方法，具有高最大容量、选择性、提取效率和稳定性的特点。
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