ORIGINAL PAPER Development and validation of a rapid method for microcystins in fish and comparing LC-MS/MS results with ELISA Lucía Geis-Asteggiante & Steven J. Lehotay & Laurie L. Fortis & George Paoli & Chandi Wijey & Horacio Heinzen Received: 6 July 2011 /Revised: 10 August 2011 /Accepted: 16 August 2011 /Published online: 1 September 2011 # Springer-Verlag (outside the USA) 2011 Abstract Microcystins (MCs) are the most common cyanotoxins found worldwide in freshwater, brackish, and marine environments. The rapid and accurate analysis of MCs and nodularin (Nod-R) in fish tissue is important for determining occurrence, following trends, and monitoring exposure for risk assessment and other purposes. The aim of this study was to develop a streamlined and reliable sample preparation method for eight MCs (MC-RR, MC- YR, MC-LR, MC-WR, MC-LA, MC-LY, MC-LW, and MC- LF) and Nod-R in fish, and conduct a validation of the new method using liquid chromatography–tandem mass spec- trometry (LC-MS/MS) for analysis and compare the results with a commercial enzyme-linked immunosorbent assay (ELISA) kit. Different sample preparation methods were compared, and a simple extraction protocol with acidified acetonitrile/water (3:1) followed by hexane partitioning cleanup was found to be most effective. Thorough validation of the final method was conducted, and 90– 115% recoveries were achieved for all analytes except for MC-RR, which gave 130% average recovery (isotopically labeled internal standards were unavailable to correct for possible biases). The use of electrospray ionization in the negative mode gave few interferences and minimal matrix effects in the LC-MS/MS analysis overall. Precision was typically 10–20% RSD among multiple days in experiments, detection limits were <10 ng/g in the fish tissue (catfish, basa, and swai filets), and no false-positives or false-negatives occurred in blind analyses of many spiked samples. The ELISA was unable to distinguish between MCs but was found to correctly assess the presence or absence of MCs and Nod-R in the blind-fortified fish tissues. The capability of these approaches to measure covalently bound MCs was not assessed. Keywords Microcystins . LC-MS/MS . ELISA . Fish tissue . Validation Introduction Microcystins (MCs) are the most common cyanotoxins found worldwide in freshwater, brackish, and marine environments [1]. Various genera of freshwater cyanobacteria, such as Microcystis, Anabaena, Oscillatoria (Planktothrix), and occasionally Nostoc, produce the toxins [2, 3]. There are over 80 MC analogues with chemical structures which all contain a cyclic heptapeptide of five amino acids common to all MCs plus two variable L-amino acids. The variable amino Mention of brand or firm name does not constitute an endorsement by the US Department of Agriculture above others of a similar nature not mentioned. Electronic supplementary material The online version of this article (doi:10.1007/s00216-011-5345-0) contains supplementary material, which is available to authorized users. L. Geis-Asteggiante : S. J. Lehotay (*) : L. L. Fortis : G. Paoli : C. Wijey Agricultural Research Service, Eastern Regional Research Center, US Department of Agriculture, 600 East Mermaid Lane, Wyndmoor, PA 19038, USA e-mail: steven.lehotay@ars.usda.gov L. Geis-Asteggiante : H. Heinzen Cátedra de Farmacognosia y Productos Naturales, DQO, Facultad de Química, UdelaR, General Flores 2124, Montevideo 12800, Uruguay Present Address: L. L. Fortis USDA National Institute of Food and Agriculture, 1400 Independence Ave., SW, Stop 2201, Washington, DC 20250, USA Anal Bioanal Chem (2011) 401:2617–2630 DOI 10.1007/s00216-011-5345-0