Submitted to: Analytical and Bioanalytical Chemistry
Publication Type: Peer reviewed journal
Publication Acceptance Date: 5/17/2011
Publication Date: 9/1/2011
Citation: Geis-Asteggiante, L., Lehotay, S.J., Fortis, L.L., Paoli, G., Wijey, C., Heinzen, H. 2011. Development and validation of a rapid method for microcystins in fish and comparing LC-MS/MS results with ELISA. Analytical and Bioanalytical Chemistry. 401:2617-2630. Interpretive Summary: Microcystins are the most common cyanotoxins found world-wide in freshwater, brackish and marine environments. The rapid and accurate analysis of microcystins (and a similar toxin, nodularin) in fish tissue is important for determining occurrence, monitoring trends, and exposure monitoring for risk assessment and other purposes. A new method of analysis that is faster, simpler, and more effective than current methods described in the scientific literature was developed and demonstrated to be useful for the analysis of the toxins in catfish and other fish. The USDA Food Safety Inspection Service requested the development of this method for its potential use in their catfish monitoring programs or the Food Emergency Response Network of laboratories.
Technical Abstract: Microcystins (MCs) are the most common cyanotoxins found world-wide in freshwater, brackish and marine environments. The rapid and accurate analysis of microcystins and nodularin in fish tissue is important for determining occurrence, monitoring trends, and exposure monitoring for risk assessment and other purposes. The aim of this study was to develop a streamlined and reliable sample preparation method for 8 MCs (-RR, -YR, -LR, -WR, -LA, -LY, -LW, and -LF) and nodularin-R (Nod-R) in fish, and conduct a side-by-side validation of the new method using commercial enzyme-linked immunosorbent assay (ELISA) and liquid chromatography – tandem mass spectrometry (LC-MS/MS) method for analysis. Different sample preparation methods were compared, and a simple extraction protocol with acidified acetontrile/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 the analytes except for MC-RR, which gave 130% average recovery (lack of an isotopically-labeled internal standard caused a high bias). The use of electrospray 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 of 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 the 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.