CYN determination in tissues from freshwater fish by LC-MS/MS: validation and application in tissues from...

Harmful cyanobacterial blooms are occurring in eutrophic freshwater lakes and reservoirs throughout the world and, because of the production of toxins such as cylindrospermopsin (CYN), they can present a public safety hazard through contamination of seafood and fish for human consumption. Therefore it is important to develop methods to determine CYN at trace levels in those organisms. A new method for unconjugated CYN determination in tissues (liver and muscle) of tilapia (Oreochromis niloticus) is herein described and discussed; it is based on solvent extraction and purification with C18 and graphitized carbon cartridges, and quantification by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The method was optimized and suitably validated, with a linear range from 0.125-12.5 µg CYN/g dry weight (dw) in the case of the liver, and 0.02-1 µg CYN/g dw for the muscle. Limits of detection and quantitation were 0.07 and 0.12 µg/g dw for the liver, and 0.002 and 0.007 µg/g dw for the muscle, respectively. Mean recoveries ranged 80-110% in liver, and 94-104% in muscle, and intermediate precision values from 6 to 11%. The method is robust against the three factors considered for purification (batch of the graphitized carbon cartridges, time for the sample to pass through the cartridge, and final dissolving water volume). Furthermore, it has been successfully applied to the extraction and quantification of CYN in tissue samples from tilapia subchronically exposed to CYN in the laboratory. This represents a sensitive, reproducible, accurate, and robust method for extraction and determination of unconjugated CYN in tissues of fish exposed to the toxin. This procedure can be used for confirmatory routine monitoring of CYN in fish samples in environmental studies.