A “Two-in-One” Tandem Immunoaffinity Column for the Sensitive and Selective Purification and Determination of Trace/Ultra-Trace Olaquindox and Its Major Metabolite in Fish Tissues by LC–MS/MS

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.