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A sensitive and selective method using high-performance liquid chromatography in combination with atmospheric pressure chemical ionization tandem mass spetrometry (LC-APCI-MS/MS) has been developed for the determination of Deoxynivalenol (DON) in trace levels. The extract was purified with a MultiSep™ column followed by the Vicam™ DON immunoaffinity column. Quantification is based on an external standard method using positive Multiple Reaction Monitoring (MRM). The limit of detection was 5 μg/kg with a signal to noise ratio of 3:1.