A “two-in-one” bilayer immunoaffinity column (IAC) coupled in tandem was prepared for the highly selective purification of olaquindox (OLA) and its major metabolite, 3-methyl-quinoxaline-2-carboxylic acid (MQCA), and their determinations were carried out with the developed high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) method. The prepared highly selective two-in-one IAC was used for fish tissue sample clean-up after acidolysis. Then, the purified samples were analyzed by LC–MS/MS in ESI⁺ and multiple reaction monitoring mode. The calibration curves showed excellent linearity, with a correlation coefficient (R²) greater than 0.999. Under the optimal conditions, the average recoveries at the spiked levels of 1.0, 5.0, and 10.0 μg/kg were 81.2–94.6% with intra- and inter-day relative standard deviations (RSD; intra-day, n = 5; inter-day, n = 3) that were less than 8.1% and 11.7%, respectively. The limits of detection (LODs) were 0.01 and 0.3 μg kg⁻¹, and the limits of quantifications (LOQs) were 0.04 and 0.5 μg kg⁻¹ for OLA and MQCA, respectively. The validated method was successfully applied to the qualitative and quantitative detection of real samples collected from several local markets.

制备了串联连接的“二合一”双层免疫亲和柱（IAC），用于高度选择性地纯化olaquindox（OLA）及其主要代谢物3-甲基-喹喔啉-2-羧酸（MQCA）和它们的使用发达的高效液相色谱-串联质谱法（LC-MS / MS）进行测定。制备的高选择性二合一IAC用于酸解后的鱼组织样品净化。然后，通过LC-MS / MS在ESI ⁺和多反应监测模式下分析纯化的样品。校准曲线显示出极好的线性，相关系数为（R ²）大于0.999。在最佳条件下，加标浓度为1.0、5.0和10.0μg/ kg时的平均回收率为81.2–94.6％，日内和日间相对标准偏差（RSD；日内，n  = 5；天，n  = 3）分别小于8.1％和11.7％。检测（检测限）限为0.01和0.3微克千克^-1，和的量化（定量限）限为0.04和0.5微克千克^-1为OLA和MQCA，分别。经过验证的方法已成功应用于从几个本地市场收集的真实样品的定性和定量检测。