Validation of an UHPLC-MS/MS method for simultaneous analysis of 11 mycotoxins in wheat flour Using Immunoaffinity column

Document Type: Research article


1 Department of Pathobiology, Science and Research Branch, Islamic Azad University, Tehran , Iran.

2 Department of Medical Parasitology and Mycology, School of Public Health, Food Microbiology Research Center, Tehran University of Medical Sciences, Tehran, Iran.

3 Department of Toxicology and Pharmacology, School of Pharmacy, Shahid Beheshti University of Medical Sciences,Tehran, Iran.


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: Aflatoxin B1, T-2 Toxin, Ochratoxin A, Deoxynivalenol and Zearalenone 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. Analysis was accepted and validated for the simultaneous quantification of 11 mycotoxins at different levels (2-150 ngg-1 for AFs, T-2 TOXIN, OTA; 20-1500 ngg-1 for ZER, HT-2 TOXIN; and 100-1500 ngg-1 for DON and FB1+B2) in wheat. Calibration curves were plotted with spiked wheat flour blank samples without internal standard. Limits of detection (LOD) ranged from 0.7 to 33.3ngg-1 and limits of quantification (LOQ) ranged from 2 to 100 ngg-1. The recoveries of selected mycotoxins were in the range of 72.2-122 %.The recoveries and repeatabilities were in accordance with the criteria determined by European Union (EU) Recommendation 519/2014.


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