ORIGINAL PAPER Mycotoxin production by pure fungal isolates analysed by means of an uhplc-ms/ms multi-mycotoxin method with possible pitfalls and solutions for patulin-producing isolates Els Van Pamel & Geertrui Vlaemynck & Marc Heyndrickx & Lieve Herman & Annemieke Verbeken & Els Daeseleire Received: 8 July 2010 / Revised: 8 October 2010 / Accepted: 8 October 2010 / Published online: 5 November 2010 # Society for Mycotoxin Research and Springer 2010 Abstract This study is the first report of applying an ultra high performance liquid chromatography/tandem mass spectrometric (UHPLC-MS/MS) multi-mycotoxin method to identify and quantify the mycotoxins produced by pure fungal isolates grown on Yeast Extract Sucrose (YES) agar. The method developed concerns a triple extraction proce- dure based on methanol, dichloromethane and ethyl acetate. The total extract was chromatographically separated on an UHPLC BEH C 18 column and analyzed with a triple quadrupole mass spectrometer. Performance characteristics (specificity, linearity, possible matrix effects, recovery, repeatability, reproducibility and limit of detection) were evaluated by spiking experiments with blank agar plugs and the analytes. Verrucarol was used as internal standard. Recovery percentages varied between 56 and 125%, whereas the limit of detection ranged from 1 to 1,500 ng g −1 with the exception of NIV, PAT and ZEA. The method was successfully applied for examining the in vitro mycotoxin production by Aspergillus fumigatus, A. flavus and A. niger. The mobile phases used for chromato- graphic separation were slightly modified when studying patulin-producing molds due to signal interference between this mycotoxin and an unknown metabolite. This modified method was successfully applied for Penicillium roqueforti, P. paneum and P. carneum grown on YES agar medium. Application of the multi-mycotoxin UHPLC-MS/MS meth- od developed may be of great importance for studying the mycotoxin capacity of fungal isolates under varying growth conditions, in order to obtain a better insight into the conditions which induce or suppress mycotoxin production by pure fungal isolates or from a chemotaxonomic point of view. Keywords Molds . Yeast extract sucrose agar . Mycotoxin production . Multi-mycotoxin analysis . UHPLC-MS/MS Introduction Mycotoxins are secondary metabolites produced by differ- ent fungal species under various conditions, either in the field or during storage. More than 150 mycotoxigenic fungi are currently known, with most belonging to the genera Fusarium, Penicillium, Aspergillus and Alternaria. Since most mycotoxigenic species can produce more than one mycotoxin, approximately 300–400 mycotoxins have been discovered up to now. Due to their high degree of structural diversity and biosynthetic origins, classifying these myco- toxins is rather challenging. The major mycotoxins are the aflatoxins, fumonisins, trichothecenes (type A and B), ochratoxins, ergot alkaloids, citrinin, patulin, and zearale- none and its metabolites (Bennet and Klich 2003). Acute effects as well as chronic effects have been reported. The high structural diversity implies that these toxins have a E. Van Pamel (*) : G. Vlaemynck : M. Heyndrickx : L. Herman : E. Daeseleire Technology and Food Science Unit, Flemish Government Institute for Agricultural and Fisheries Research (ILVO), Brusselsesteenweg 370, 9090 Melle, Belgium e-mail: els.VanPamel@ilvo.vlaanderen.be A. Verbeken Department of Biology, Research Group Mycology, Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Mycotox Res (2011) 27:37–47 DOI 10.1007/s12550-010-0073-4