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.