Identification of microcystins in a Lake Victoria cyanobacterial bloom using LC–MS with thiol derivatization

Abstract

Microcystins are cyclic heptapeptides from cyanobacteria which are responsible for poisonings of livestock and humans. Cyanobacteria also produce a range of peptides and other compounds that can result in complex chromatograms when samples are analysed by LC–MS. Thiol derivatization of the α,β-unsaturated amide present in most microcystins was recently shown to simplify analysis of LC–MS chromatograms of a Microcystis culture, making it easier to identify peaks corresponding to microcystins in complex mixtures. This method was applied to analysis of extracts taken from a natural cyanobacteria bloom in Mwanza Gulf, Lake Victoria, Tanzania, in 2010, revealing the presence of numerous putative microcystin analogues in the sample. Results were verified using LC–MS², LC–MS/MS with precursor-ion scanning, and LC–HRMS, leading to identification of 8 major and 17 minor microcystins in the sample, including analogues of microcystin-RY, -RL and -RA. Microcystin-YR (2), -RR (3), and -RY (9) were isolated from bloom material from Lake Victoria, and the structure of 9 was confirmed by NMR spectroscopic analysis and NMR spectral comparison with 2 and 3. Confirmation of the structure of MC-RY (9) facilitated detailed analysis of its MS² spectrum, thereby supporting the structures of related analogues tentatively established on the basis of MS analyses.