Intracellular cyanotoxins were extracted from filters applying the protocol described by Cerasino and Salmaso [48 (link)] and quantified with LC-MS/MS. The extraction was carried out by using a mixture of acetonitrile in water (60/40 v/v), containing 0.1% formic acid. Extracted toxins were injected into a LC-MS/MS system, composed of a Waters Acquity UPLC system (Waters, Milford, MA, USA) coupled to a SCIEX 4000 QTRAP mass spectrometer (AB Sciex Pte. Ltd., Singapore). The mass detector was operated in scheduled MRM (Multiple Reaction Monitor) mode, using positive electrospray ionisation (ESI+). Quantification of microcystins was performed following the protocol from Cerasino and Salmaso [48 (link)], which has the capability of determining the 11 most common microcystin variants, namely RR, [D-Asp3]-RR (RRdm), [D-Asp3]-HtyrR (HtyRdm), YR, LR, [D-Asp3]-LR (LRdm), WR, LA, LY, LW, LF. Analysis of cylindrospermopsins and saxitoxins was performed following the protocol from Ballot et al. [49 (link)], targeting CYN, STX, dcSTX, NeoSTX, GTX1, GTX4, GTX5, C1 and C2.
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