A high throughput targeted and non-targeted method for the analysis of microcystins and anatoxin-A using on-line solid phase extraction coupled to liquid chromatography–quadrupole time-of-flight high resolution mass spectrometry

Microcystins are cyclic heptapeptide hepatotoxins produced by cyanobacteria in freshwater. Sample preparation for the analysis of these cyanotoxins in water from algal blooms can take up to several days due to the matrix complexity and the low detection limits required to comply with current legislation. Moreover, there is a large number of unknown microcystins that could potentially exist in the environment resulting from different amino acid substitutions into the microcystin skeletal structure. To tackle these problems, the present study involved the development of a high throughput method based on on-line solid phase extraction coupled to liquid chromatography that could provide quantitative results for 12 microcystin variants (LR, YR, RR, HtyR, HilR, WR, LW, LA, LF, LY, Dha⁷-LR, and Dha⁷-RR) and anatoxin-A in less than 3 h with detection limits between 0.004 and 0.01 μg L⁻¹ and expanded uncertainty between 4 and 14%. Data-dependent acquisition was employed for the non-targeted analysis of these cyanotoxins. Filtering the data based on structure diagnostic fragments, two unknown microcystin variants not previously reported in the literature were detected. The structures Leu¹-microcystin-Met(O)R and Leu¹-microcystin-LY were fully characterized by accurate mass measurement, collision-induced dissociation, and fragmentation prediction software.