Standardization of microcystin extraction from fish tissues: A novel internal standard as a surrogate for polar and non-polar variants

Microcystins (MCs), a class of potent liver/hepatopancreatic toxins produced by numerous species of freshwater cyanobacteria, are well known for their toxic effects on aquatic organisms and humans. The extraction efficiencies of MCs can vary greatly as a result of matrix differences and/or differences in the extraction solvents, techniques, and clean-up steps utilized. Here we report the preparation of a unique internal standard, (S-hydroxypropyl-cys7)microcystin-LR (thiol-LR), with a mass different than any known MCs, which can be spiked into field samples and quantified via HPLC-MS along with endogenous MCs. Thiol-LR is modified at the Mdha residue, and therefore, provides an accurate measure of only free MCs that are not covalently bound to endogenous thiols in the matrix. The internal standard proved to be a good surrogate for MC-RR, -LR, and -LA in fish liver tissue. In fish muscle tissue, thiol-LR was a good surrogate for polar variants, MC-RR and -LR, but was less representative of the non-polar variant, MC-LA. Coupling of the internal standard (8 μg/g ww tissue) with HPLC-MS detection will standardize the quantification of free microcystins across species, tissue types, and extraction methods, making the estimation of exposure risk more reliable.