Analysis of multiple mycotoxins in beer employing (ultra)-high-resolution mass spectrometry Milena Zachariasova 1 , Tomas Cajka 1 , Michal Godula 2 , Alexandra Malachova 1 , Zdenka Veprikova 1 and Jana Hajslova 1 * 1 Institute of Chemical Technology, Prague, Faculty of Food and Biochemical Technology, Department of Food Chemistry and Analysis, Technicka 3, 166 28 Prague 6, Czech Republic 2 Thermo Fisher Scientific, Czech Republic, Slunecna 27, 100 00 Prague 10, Czech Republic Received 6 May 2010; Revised 17 August 2010; Accepted 22 August 2010 The objective of the presented study was to develop and optimize a simple, high-throughput method for the control of 32 mycotoxins (Fusarium and Alternaria toxins, aflatoxins, ergot alkaloids, ochratoxins, and sterigmatocystin) in beer. Due to the broad range of their physicochemical properties, the sample preparation step was simplified as much as possible to avoid analyte losses. The addition of acetonitrile to beer samples enabled precipitation of abundant matrix components. The clean-up efficiency was controlled by ambient mass spectrometry employing a direct analysis in real time (DART) ion source. For determination of analytes, ultra-high-performance liquid chromatography hyphenated with high-resolution mass spectrometry utilizing an orbitrap (U-HPLC–orbitrapMS) or time-of-flight (TOFMS) technology was used. Because of significantly better detection capabilities of the orbitrap technology, the U-HPLC–orbitrapMS method was chosen as a determinative step and fully validated. To compensate matrix effects, matrix-matched calibration was employed. The lowest calibration levels for most of the target mycotoxins ranged from 1 to 8 mgL 1 beer and the recoveries of analytes were in range from 86 to 124%. Copyright # 2010 John Wiley & Sons, Ltd. Mycotoxins are toxic secondary metabolites produced by many species of microscopic filamentous fungi occurring on field cereals, including barley. The most abundant fungal genera affecting the malting barley are Alternaria, Aspergillus, Penicillium, and Fusarium, which simultaneously showed relatively high producing potential for a wide range of mycotoxins (almost 30% of Alternaria, 20% of Aspergillus, and 88% of Fusarium fungi isolated from barley grains was able to produce alternaria toxins, aflatoxins, ochratoxin A, deoxynivalenol and zearalenone). 1 Additionally to the relatively common micromycetes mentioned above, also the Claviceps purpurea causing the ergot disease belongs to frequent barley pathogens. 2 Although the carry-over of aflatoxins, ochratoxin A, zearalenone, fumonisins, and ergot alkaloids from malted grains into beer has been documented, 2,3 the main research in this area has been focused on deoxynivalenol, the most frequent Fusarium mycotoxin. 4–8 In recent years, the presence of its main metabolite, deoxynivalenol-3-glucoside, in malt and beer has been reported at relatively high levels (the deoxynivalenol-3-glucoside/deoxynivalenol molar ratio was mostly even 1). 9 This was further confirmed in our follow- up study, in which both deoxynivalenol and its glucoside were determined as the main contaminants of beers retailed on the European market. 10 Since beer significantly contrib- utes to the diet of a population, control of the presence of mycotoxins in this commodity is very important. For this purpose, reliable analytical methods for fast and effective monitoring of mycotoxins in the beer production chain are needed. Over the years, several multidetection methods for the determination of mycotoxins in beer have been developed (an overview is summarised in Table 1). The first of them employed the gas chromatographic (GC) approach with derivatisation of analytes, 11 which is nowadays practically out of the use. All of the later published methods have already been based on liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS); 12–15 however, employing a more-or-less specific clean-up step limits the number of analytes it is possible to determine within the analysis. Nowadays, there is a trend towards simplifying the sample preparation procedure as much as possible, and, simultaneously, the full spectral data acquisition techniques are preferred because of their ease of use, and the possibility of retrospective mining of archived data. Until recently, the most common full spectral MS approach has been time-of- flight (TOF)MS, with a typical mass resolving power of benchtop instruments of 10 000–12 000 FWHM (full width at half maximum). However, in complex food matrices such as beer, this rather limited mass resolving power leads to the risk of inaccurate mass measurements caused by unresolved background matrix interferences. 16,17 Contrary to that, recently introduced benchtop systems based on the orbitrap technology, a special type of ion trap with a central spindle- shaped electrode around which the trapped ions oscillate, RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2010; 24: 3357–3367 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/rcm.4746 *Correspondence to: J. Hajslova, Institute of Chemical Technology, Prague, Faculty of Food and Biochemical Technology, Depart- ment of Food Chemistry and Analysis, Technicka 3, 166 28 Prague 6, Czech Republic. E-mail: jana.hajslova@vscht.cz Copyright # 2010 John Wiley & Sons, Ltd.