Analytical and Bioanalytical Chemistry

, Volume 401, Issue 8, pp 2617–2630 | Cite as

Development and validation of a rapid method for microcystins in fish and comparing LC-MS/MS results with ELISA

  • Lucía Geis-Asteggiante
  • Steven J. LehotayEmail author
  • Laurie L. Fortis
  • George Paoli
  • Chandi Wijey
  • Horacio Heinzen
Original Paper


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 spectrometry (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.


Microcystins LC-MS/MS ELISA Fish tissue Validation 



We thank Jennifer Cassidy and Kathleen Rajkowski for providing the different fish samples, Alan Lightfield for his support in LC-MS/MS usage, and Fernando Rubio of Abraxis for consultation about microcystins and ELISA. This research was funded in part by the USDA Food Safety Inspection Service ARS agreement number 60-1935-9-031 and the US–Israel Binational Agricultural Research and Development Fund number US-4273-09.

Supplementary material

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Supplementary material, approximately 509 KB. (7 mb)
Supplementary material, approximately 7.02 MB.


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Copyright information

© Springer-Verlag (outside the USA) 2011

Authors and Affiliations

  • Lucía Geis-Asteggiante
    • 1
    • 2
  • Steven J. Lehotay
    • 1
    Email author
  • Laurie L. Fortis
    • 1
    • 3
  • George Paoli
    • 1
  • Chandi Wijey
    • 1
  • Horacio Heinzen
    • 2
  1. 1.Agricultural Research Service, Eastern Regional Research CenterUS Department of AgricultureWyndmoorUSA
  2. 2.Cátedra de Farmacognosia y Productos Naturales, DQO, Facultad de QuímicaUdelaRMontevideoUruguay
  3. 3.USDA National Institute of Food and AgricultureWashingtonUSA

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