Comprehensive IAC Cross-Reactivity Validation and Stabilized Method Development for Ochratoxin A, B, and C in Complex Coffee and Spice Matrices

Ochratoxins (OTs) pose a major food safety threat, yet analytical methodologies and regulations focus almost exclusively on ochratoxin A (OTA), overlooking the toxic analogues OTB and OTC, especially in complex coffee and spice matrices. The present study addressed this gap by first systematically confirming the high cross-reactivity (>85%) of commercial OTA immunoaffinity columns (IACs) toward OTB and OTC. It was identified that conventional alkaline methanol extraction caused OTC degradation, and subsequently a stable and unified acetonitrile-water (8/2, v/v) extraction protocol was developed. To overcome severe matrix interference endemic to these foods, a novel 0.5% Tween-20-PBS IAC load and wash procedure was optimized. The resulting method was fully validated in representative roasted coffee and pepper matrices on both HPLC-FLD and UHPLC-MS/MS platforms, demonstrating excellent linearity (r > 0.999), accuracy (mean recovery 82.00–112.51%), and precision (RSD% ≤ 8.81%) across three spiked levels (0.3, 5, 10 µg/kg). While UHPLC-MS/MS achieved higher sensitivity (LOQs 0.1 µg/kg) than that of HPLC-FLD (LOQs 0.3 µg/kg), with isotope internal standards essential for correcting significant matrix effects. Application to forty commercial coffee and spice samples (19 coffee, 21 spice) revealed OTA contamination in 47.5% of products (up to 3.46 µg/kg) and co-occurrence of OTA/OTB in 3 of 8 cumin samples. This work establishes the first comprehensively validated IAC-based method for multi-OTs in complex foods, facilitating an urgently needed, robust tool for comprehensive risk assessment.