A validated UPLC–MS/MS method for the surveillance of ten aquatic biotoxins in European brackish and freshwater systems Brett Greer a , Sara E. McNamee a , Bas Boots b , Lucia Cimarelli c , Delphine Guillebault d , Karim Helmi e , Stefania Marcheggiani f , Stefan Panaiotov g , Ulrich Breitenbach h , Reyhan Akc ¸aalan i , Linda K. Medlin d , Katrin Kittler j , Christopher T. Elliott a , Katrina Campbell a, * a Institute for Global Food Security, School of Biological Sciences, Queen’s University Belfast, Stranmillis Road, Belfast BT9 5AG, UK b University College Dublin, National University of Ireland, Dublin, Ireland c Laboratory of Genetics, School of Biosciences and Veterinary Medicine, University of Camerino, Camerino, MC 62032, Italy d Universite Pierre et Marie Curie, Paris 6, France e Veolia Environnement Recherche et Innovation SNC, France f Istituto Superiore di Sanita, Rome, Italy g National Center of Infectious and Parasitic Diseases, Sofia 1504, Bulgaria h Marilim Gesellschaft fur Gewasseruntersuchung mbH, Germany i Istanbul University, Istanbul, Turkey j BAM Federal Institute for Materials Research and Testing, Richard-Willsta ¨tter-Straße 11, 12489 Berlin, Germany 1. Introduction Cyanobacteria (blue-green algae) are photosynthetic bacteria which have been present on earth for several billion years. They are found globally in aquatic environments such as lakes, rivers, ponds, reservoirs and brackish waters (Codd et al., 2005), as well as in terrestrial and aerial environments (Metcalf et al., 2012). Some cyanobacteria are known to produce secondary metabolites called Harmful Algae 55 (2016) 31–40 A R T I C L E I N F O Article history: Received 30 August 2015 Received in revised form 14 January 2016 Accepted 14 January 2016 Available online 12 February 2016 Keywords: Harmful Algal Bloom (HAB) Biotoxins Multiplex UPLC–MS/MS Microcystin A B S T R A C T Over the past few decades, there has been an increased frequency and duration of cyanobacterial Harmful Algal Blooms (HABs) in freshwater systems globally. These can produce secondary metabolites called cyanotoxins, many of which are hepatotoxins, raising concerns about repeated exposure through ingestion of contaminated drinking water or food or through recreational activities such as bathing/ swimming. An ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) multi-toxin method has been developed and validated for freshwater cyanotoxins; microcystins-LR, -YR, -RR, -LA, -LY and -LF, nodularin, cylindrospermopsin, anatoxin-a and the marine diatom toxin domoic acid. Separation was achieved in around 9 min and dual SPE was incorporated providing detection limits of between 0.3 and 5.6 ng/L of original sample. Intra- and inter-day precision analysis showed relative standard deviations (RSD) of 1.2–9.6% and 1.3–12.0% respectively. The method was applied to the analysis of aquatic samples (n = 206) from six European countries. The main class detected were the hepatotoxins; microcystin-YR (n = 22), cylindrospermopsin (n = 25), microcystin-RR (n = 17), micro- cystin-LR (n = 12), microcystin-LY (n = 1), microcystin-LF (n = 1) and nodularin (n = 5). For microcystins, the levels detected ranged from 0.001 to 1.51 mg/L, with two samples showing combined levels above the guideline set by the WHO of 1 mg/L for microcystin-LR. Several samples presented with multiple toxins indicating the potential for synergistic effects and possibly enhanced toxicity. This is the first published pan European survey of freshwater bodies for multiple biotoxins, including two identified for the first time; cylindrospermopsin in Ireland and nodularin in Germany, presenting further incentives for improved monitoring and development of strategies to mitigate human exposure. ß 2016 Elsevier B.V. All rights reserved. * Corresponding author at: Institute for Global Food Security, School of Biological Sciences, Queen’s University, David Keir Building, Stranmillis Road, Belfast BT9 5AG, UK. Tel.: +44 02890976535; fax: +44 02890976513. E-mail address: katrina.campbell@qub.ac.uk (K. Campbell). Contents lists available at ScienceDirect Harmful Algae jo u rn al h om epag e: ww w.els evier.c o m/lo cat e/hal http://dx.doi.org/10.1016/j.hal.2016.01.006 1568-9883/ß 2016 Elsevier B.V. All rights reserved.