免疫亲和柱净化-高效液相色谱-可见检测法测定鱼肉中孔雀石绿、结晶紫及其无色代谢产物
Determination of malachite green, crystal violet and their leuco-metabolites in fish by HPLC-VIS detection after immunoaffinity column clean-up.
机构信息
Department of Chemistry and Chemical Engineering, University of South China, Hunan, Hengyang 421001, China.
出版信息
J Chromatogr B Analyt Technol Biomed Life Sci. 2013 Jan 15;913-914:123-8. doi: 10.1016/j.jchromb.2012.12.002. Epub 2012 Dec 10.
A high performance liquid chromatography method with visible detection (HPLC-VIS) for the determination of malachite green (MG), crystal violet (CV), leucomalachite green (LMG), and leucocrystal violet (LCV) in fish has been developed after clean-up through an immunoaffinity column (IAC). Residues were simultaneously extracted from fish muscle with acetonitrile and ammonium acetate buffer. The leuco-forms, LMG and LCV, were oxidized quantitatively to the chromic CV and MG by reaction with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone. Extracts were then purified on an IAC which prepared by immobilizing the anti-MG-CV antibodies by the sol-gel method. Finally, the eluents were analyzed by HPLC-VIS. The limits of detection were 0.15, 0.1, 0.18 and 0.14ng/g for MG, CV, LMG and LCV, respectively. The average recoveries in samples fortified with MG, CV, LMG and LCV over the range 0.5-10ng/g were from 71.6% to 96.8% with RSDs of 5.1-12.3% (n=6). This novel method was confirmed by liquid chromatography-tandem mass spectrometry with electrospray interface in positive mode using multiple reaction monitoring.
建立了一种免疫亲和色谱（IAC）净化-可见高效液相色谱法（HPLC-VIS），用于测定鱼中孔雀石绿（MG）、结晶紫（CV）、隐色孔雀石绿（LMG）和隐色结晶紫（LCV）的残留。鱼肌肉中的残留物用乙腈和乙酸铵缓冲液提取。通过与 2,3-二氯-5,6-二氰基-1,4-苯醌反应，将 LMG 和 LCV 的无色形式定量氧化为有色的 CV 和 MG。然后，将提取物通过 sol-gel 方法固定抗 MG-CV 抗体制备的 IAC 进行纯化。最后，用 HPLC-VIS 分析洗脱液。MG、CV、LMG 和 LCV 的检测限分别为 0.15、0.1、0.18 和 0.14ng/g。在 0.5-10ng/g 的范围内，用该方法对 MG、CV、LMG 和 LCV 进行加标回收试验，平均回收率为 71.6%-96.8%，相对标准偏差为 5.1%-12.3%（n=6）。该方法通过正模式电喷雾接口的液相色谱-串联质谱法（LC-MS/MS）得到了验证，采用多反应监测（MRM）。
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