Determination of microcystins in blue-green algae, fish and water using liquid chromatography with ultraviolet detection after sample clean-up employing immunoaffinity chromatography

Anti-microcystin LR immunoaffinity cartridges were evaluated for their ability to selectively remove microcystins from extracts of blue-green algae, fish and water samples for subsequent analysis by liquid chromatography with UV absorbance detection at 238 nm. Blue-green algae and fish samples were extracted with 75% methanol in water. A portion of the extract was diluted and passed through an immunoaffinity cartridge. Water samples were applied directly to the cartridge. The cartridge was rinsed with water and 25% methanol in water. The microcystins were eluted with 80% methanol in water containing 4% acetic acid. It was found that the cartridges were effective in isolating the microcystins from blue-green algae, fish and water samples, resulting in extracts that were clean enough to enable direct LC–UV detection down to ∼0.03 μg/g in the blue-green algae and fish samples, and as low as 0.02 ng/ml for water samples. The cartridges were found to have a capacity of ∼200 ng each for a mixture of microcystins RR, YR, LR and LA, or as much as 525–800 ng for individual compounds. Recoveries through the complete analytical procedure ranged from 64 to 115% (all values) with an overall average of ∼80% at spiking levels of 0.5–4.0 μg/g for the microcystins in blue-green algae. The average recoveries (n=8) from spiked (0.1–0.5 μg/g) fish samples were 73% for RR, 79% for YR, 81% for LR and 77% for LA, while from the spiked (2.0–0.04 ng/g) tap and river water samples (n=6), recoveries were 78% for RR, 86% for YR, 94% for LR and 89% for LA.