Development and Validation of an HPLC-MS/MS Method for Quantifying Deoxynivalenol and Zearalenone Biomarkers in Dried Porcine Blood Spots

Deoxynivalenol (DON) and zearalenone (ZEN) are common mycotoxins in animal feeds, and their metabolites can be detected in exposed animals. Traditional methods focus on mycotoxin detection in feed, whereas biomarker-based approaches are used for evaluating individual exposure. This study aimed to develop and validate a multi-analyte method for the detection of biomarkers of ZEN, DON, and their metabolites α-zearalanol (α-ZAL), zearalanone (ZAN), deepoxy-DON (DOM-1), and 3-acetyl-DON (3-ADON) in swine using dried blood spots (DBSs) on qualitative filter paper. Analysis was performed using high-performance liquid chromatography–tandem mass spectrometry. Blank blood samples from three male pigs were fortified with 20, 40, and 60 μg/L of each analyte. Aliquots of 40 μL were spotted onto filter paper and then extracted and analyzed. Method validation included evaluating limits of detection and quantification, linearity, matrix effects, recovery, repeatability, intermediate precision, and selectivity. All analytes were detectable in DBS. Also, ZEN, ZAN, DON, and DOM-1 met all validation criteria, with recovery values of 89.10%, 79.79%, 101.50%, and 79.50%, respectively. Both α-ZAL and 3-ADON showed lower recoveries (74.66% and 58.66%). The method was successfully validated for simultaneous analysis of ZEN, ZAN, DON, and DOM-1 in swine DBS, offering a practical and minimally invasive tool for biomonitoring mycotoxin exposure.