Accurate LC-MS analyses for microcystins using per-15N-labeled microcystins

摘要

Per-15N-labeled microcystins were prepared for use as surrogates for accurate liquid chromatography–mass spectrometry analysis. Two strains of Microcystis aeruginosa were cultured in 15NO₃-containing TS-15 medium. To change from the incorporation of 14N to 15N into all cell components, cells of Microcystis aeruginosa were precultured in Na15NO₃-containing medium for more than 6 months. After mass cultivation of the strains, cells of each strain were harvested and lyophilized. Microcystin variants were extracted from the lyophilized cells and per-15N-labeled microcystin variants were purified using high-performance liquid chromatography and high-performance thin-layer chromatography. The structures of per-15N-labeled microcystin variants were confirmed by their mass spectrometry spectra and NMR spectra. When per-15N-labeled microcystins were used as surrogates for quantitative analysis of these toxins in cyanobacterial cells, excellent accuracy (98–106%) was obtained, with the m/z of M+, [M+1]+, and [M+2]+ of both microcystins and the per-15N-labeled microcystins as surrogates being completely separated. In conclusion, per-15N-labeled microcystins are excellent surrogates for microcystin analysis using liquid chromatography–mass spectrometry.