Determination of microcystins in biological samples from freshwater fish

Abstract

A new HPLC–DAD method has been developed to identify and quantify free microcystins (MC) in biological samples from fish (intestine and liver). The toxins were extracted from 500 mg sample with a mixture of methanol–water (85 : 15, v/v) and the extracts obtained were purified employing immunoaffinity columns (IAC). The purification step was optimised by a full factorial 3² design. MC were separated using conventional C18 column and an acetonitrile-acidified water (pH 3) gradient. Detection and quantification limits resulted equal for the two toxins assayed (MC-RR and MC-LR) and were 0.15 and 0.5 µg g⁻¹, respectively. The accuracy for each MC in liver samples were 96% (range 80–113%) for MC-RR and 101% (range 93–118%) for MC-LR. The results were slightly lower for intestine samples, with recoveries ranging between 85% (75–93%) for MC-RR and 88% (80–97%) for MC-LR. The proposed method was applied for the determination of free MC in fish intoxicated with these toxins, in order to determine its utility to evaluate the potential risks for human health if MC-contaminated fish are consumed. The results showed the transference of MC-LR from cyanobacterial cells to fish tissues.

Keywords:

• microcystins
• purification
• immunoaffinity column
• liver
• intestine
• fish
• liquid chromatography–DA

Acknowledgements

The authors wish to thank the CICYT (AGL 2006-06523/ALI) for the financial support for this study.