Validation of an UHPLC-MS/MS Method for Simultaneous Analysis of 11 Mycotoxins in Wheat Flour Using Immunoaffinity Column

This study focuses on optimization and validation of an Ultrahigh-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous analysis of 11mycotoxins: Aflatoxins (B(1), B(2), G(1), and G(2)), Ochratoxin A, Deoxynivalenol, Fumonisins (B(1) and B(2)), Zearalenone, T-2, and HT-2toxin, in wheat matrix. Sample extraction and cleanup procedure is based on a single extraction step using acetonitrile/water/acetic acid mixture (79.5/20/0.5 v/v/v) and rapid clean-up of samples were performed with the Myco6in1(+) Immunoaffinity column. Electrospray ionization at positive mode was operated to the simultaneously analysis of selected mycotoxins in a single run time of 15 min. Multiple Reaction Monitoring (MRM) mode was selected for quantification and detection of the mycotoxins. The analysis method was validated for selected mycotoxins at different spike levels (2-150 ngg(-1) for AFs, T-2, OTA; 20-1500 ngg(-1) for ZER, HT-2 toxin; and 100-1500 ngg(-1) for DON and FB(1)+B(2)) in wheat. Calibration curves were plotted based on the area of peak analyte in spike samples. Limits of detection (LOD) ranged from 0.7 to 33.3 ngg(-1) and limits of quantification (LOQ) ranged from 2 to 100 ngg(-1). Recovery values were between 70 and 120% for all the mycotoxins, except for AFG(2 )(72-123%) and T-2 toxin (77-122%) with good repeatability. The recoveries and repeatabilities were in accordance with the criteria determined by European Union (EU) Recommendation 519/2014.