Key Laboratory of Green Chemistry & Technology, Ministry of Education of China, College of Chemistry, Sichuan University, Chengdu 610064, China.
Talanta. 2009 Nov 15;80(1):98-103. doi: 10.1016/j.talanta.2009.06.029. Epub 2009 Jun 18.
The preparation and characterization of an immunoaffinity chromatography (IAC) column for the specific extraction and enrichment of trace contraceptive drug levonorgestrel (LNG) from water samples were described. The IAC column was constructed by covalently coupling specific polyclonal antibody against LNG to CNBr-activated Sepharose 4B and packed into a common solid phase extraction (SPE) cartridge. The extraction conditions including loading, washing and eluting solutions, as well as the effect of flow rate on the extraction were carefully optimized. Pure water, 5% of methanol and 50% of methanol were respectively selected as loading, washing and eluting solutions, while the flow rates in the loading, washing and eluting steps were selected to be 1.0, 2.0 and 0.5 mL min(-1), respectively. Under optimal conditions, the IAC column was characterized in terms of maximum capacity, extraction recovery and stability. It was found that, for IAC column packed with 0.2g of solid support immobilized with antibody, the maximum capacity for LNG was about 260 ng. The extraction recoveries of the column for LNG at three different spiked concentrations were within 95.3-106.9%. After more than 35 times repeated usage, there was not significant loss of specific recognition. Using high performance liquid chromatography (HPLC) as an analytical tool, trace amount of LNG in the range of ng L(-1) was found in river water and wastewater samples after 600-fold enrichment, demonstrated the feasibility of the prepared IAC column for LNG extraction.
本文描述了一种免疫亲和色谱（IAC）柱的制备和特性，用于从水样中特异性提取和富集痕量避孕药左炔诺孕酮（LNG）。IAC 柱通过将针对 LNG 的特异性多克隆抗体共价偶联到 CNBr 活化的 Sepharose 4B 上构建，并填充到常见的固相萃取（SPE）小柱中。仔细优化了提取条件，包括上样、洗涤和洗脱溶液，以及流速对提取的影响。分别选择纯水、5%甲醇和 50%甲醇作为上样、洗涤和洗脱溶液，而在上样、洗涤和洗脱步骤中的流速分别选择为 1.0、2.0 和 0.5 mL min(-1)。在最佳条件下，对 IAC 柱的最大容量、提取回收率和稳定性进行了表征。结果表明，对于用 0.2g 固定化抗体的固相载体填充的 IAC 柱，LNG 的最大容量约为 260ng。该柱对 LNG 在三个不同浓度加标水平下的提取回收率在 95.3-106.9%之间。经过 35 次以上重复使用后，特异性识别能力没有明显损失。使用高效液相色谱（HPLC）作为分析工具，在经过 600 倍富集后，在河水和废水样品中发现了痕量 LNG（ng L(-1)），证明了制备的 IAC 柱用于 LNG 提取的可行性。
