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 (R-2) greater than 0.999. Under the optimal conditions, the average recoveries at the spiked levels of 1.0, 5.0, and 10.0 mu g/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 mu g kg(-1), and the limits of quantifications (LOQs) were 0.04 and 0.5 mu g 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.