Holistic approach based on high resolution and multiple stage mass spectrometry to investigate ergot alkaloids in cereals Natalia Arroyo-Manzanares a,1 , Svetlana V. Malysheva b,1 , Julie Vanden Bussche c , Lynn Vanhaecke c , José Diana Di Mavungu b,n , Sarah De Saeger b a Department of Analytical Chemistry, Faculty of Sciences, University of Granada, Campus Fuentenueva s/n, E-18071 Granada, Spain b Laboratory of Food Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium c Laboratory of Chemical Analysis, Department of Veterinary Public Health and Food Safety, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium article info Article history: Received 3 May 2013 Received in revised form 30 September 2013 Accepted 2 October 2013 Available online 15 October 2013 Keywords: Ergot alkaloid identification Untargeted analysis Orbitrap Fragmentation pathway abstract A holistic approach based on high resolution and multiple stage mass spectrometry was developed for identification of less studied or novel ergot alkaloid derivatives. Initially, the fragmentation of nine known ergot alkaloids was studied to establish a strategy for the identification of novel ergot alkaloids. Ions with m/z 223 and m/z 251 were found to be common for all ergopeptines, ergoamides and ergopeptams. Subsequently, parent scan experiments using these ions were performed to screen grain samples for the presence of possible ergot alkaloid derivatives. Besides the six most common ergot alkaloids and their corresponding epimers (for which reference standards were available), eleven other ergot alkaloid derivatives were identified following the proposed strategy. & 2013 Elsevier B.V. All rights reserved. 1. Introduction Ergot alkaloids are secondary metabolites produced by Claviceps spp., which mostly infect grains and grasses [1,2]. Ingestion of food and feed contaminated with ergot alkaloids might cause adverse health effects in humans and animals (e.g., ergotism). The poisoning is characterized by such symptoms as abdominal pain, vomiting, burning sensation of the skin, insomnia and hallucinations [3]. The majority of ergot alkaloids are commonly comprised of ergoamides, ergopeptines (or ergopeptides), also called cyclol ergot alkaloids, and the lactam ergot alkaloids, also named ergopeptams [1,4]. The ergoamides are D-lysergic acid amides (Fig. 1a), whereas the ergopeptines are D-lysergic acid peptides containing lysergic acid and three amino acids in their structure (Fig. 1b). The ergopeptams are tripeptidic non-cyclol ergot alkaloids (Fig. 1c). Their structure is similar to that of ergopeptines except that L-proline is exchanged by D-proline, and the tripeptide chain is a non-cyclol lactam [1]. To date, more than 40 ergot alkaloids are known. Several analytical techniques have been used to study the fragmentation of these compounds and attempts have been made to identify novel derivatives. In the last years, a number of new ergot alkaloids has been discovered [5–8]. Mohamed et al. [9] used triple quadrupole and multiple stage mass spectrometry (MS) to characterize six ergot alkaloids belonging to lysergic acid and peptide-type derivatives and could confirm the presence of ergosine in a rye flour extract at trace levels. Lehner et al. [7,10] using high-performance liquid chromatography (HPLC) coupled to tandem quadrupole MS and ion trap MS were able to establish the fragmentation patterns of eight ergot alkaloids and subsequently elucidate a new ergot alkaloid-related compound. Uhlig and Petersen [11] obtained structural information of four ergopeptams using LC-ion trap MS. In the above-mentioned studies, fragment assignment was supported by hydrogen/deuterium exchange [9] and/or compar- ison of the fragmentation behavior of known ergot alkaloids, using unit mass resolution data acquired by triple quadrupole and ion trap instruments. Along with fragmentation trees, accurate mass measurement is a highly important feature for correct structure elucidation [12]. Orbitrap MS is becoming a more and more popular platform for identification purposes in natural product analysis [13]. Regarding the secondary fungal metabolites and more specifically ergot alkaloids, this type of MS has been utilized only in targeted analysis [14] or in pre-selected screening using a Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/talanta Talanta 0039-9140/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.talanta.2013.10.002 n Corresponding author. Tel.: þ32 9 264 81 33; fax: þ32 9 264 81 99. E-mail addresses: narroyo@ugr.es (N. Arroyo-Manzanares), svetlana.vl.malysheva@gmail.com (S.V. Malysheva), Julie.VandenBussche@UGent.be (J. Vanden Bussche), Lynn.Vanhaecke@UGent.be (L. Vanhaecke), Jose.DianaDiMavungu@UGent.be (J. Diana Di Mavungu), Sarah.DeSaeger@UGent.be (S. De Saeger). 1 These authors contributed equally to this work. Talanta 118 (2014) 359–367