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The occurrence of mycotoxins in agricultural commodities is a major health concern for livestock, humans, and the environment. Barley and subsequently malt quality is of fundamental importance to obtain good quality beer. Classical methods of analysis often require tedious, laborious, and expensive processes. Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) instrumentation enables highly sensitive and fast analysis and/or detection using a very small sample. The possibilities of MALDI-TOF MS for he identification and/or detection of trichothecene mycotoxins (deoxynivalenol (DON) and nivalenol (NIV), respectively) in barley malt were examined, and it was found that almost all classical MALDI matrices failed to ionize the compounds being studied. This detailed study of the ionization conditions and the search for unconventional matrices led to the discovery of suitable MALDI matrices, which enable ionization of trichothecene mycotoxins. These were: fine powdered synthetic diamond, sodium azide, or hydrazine hydrate. It is possible to detect 8.5 × 10-12 mol (2.5 ng) of deoxynivalenol or 64 × 10-12 mol (20 ng) of nivalenol in just 1 μL of barley malt extract (equivalent to 600 μg of DON in 1 kg of barley malt). The procedure developed enables fast determination of DON and NIV in barley, malt, or similar products. [ABSTRACT FROM AUTHOR]