An integrated strategy for rapid and accurate determination of free and cell-bound microcystins and related peptides in natural blooms by liquid chromatography–electrospray-high resolution mass spectrometry and matrix-assisted laser desorption/ionization

• A HRMS integrated approach for free plus cell-bound MC analysis was presented.
• A non-target methodology able to retrospective analysis of any toxin was proposed.
• The composed MS strategy combines capabilities of HR, AMM and fragmentation studies.
• MALDI-TOF/TOF and Orbitrap MS methods allow rapid analysis of solid biomass and water.
• High identity assurance was obtained combining positive and negative ionization modes.

An integrated high resolution mass spectrometry (HRMS) strategy has been developed for rapid and accurate determination of free and cell-bound microcystins (MCs) and related peptides in water blooms. The natural samples (water and algae) were filtered for independent analysis of aqueous and sestonic fractions. These fractions were analyzed by MALDI-TOF/TOF-MS and ESI-Orbitrap-HCD-MS. MALDI, ESI and the study of fragmentation sequences have been provided crucial structural information. The potential of combined positive and negative ionization modes, full scan and fragmentation acquisition modes (TOF/TOF and HCD) by HRMS and high resolution and accurate mass was investigated in order to allow unequivocal determination of MCs. Besides, a reliable quantitation has been possible by HRMS. This composition helped to decrease the probability of false positives and negatives, as alternative to commonly used LC–ESI-MS/MS methods. The analysis was non-target, therefore covered the possibility to analyze all MC analogs concurrently without any pre-selection of target MC. Furthermore, archived data was subjected to retrospective “post-targeted” analysis and a screening of other potential toxins and related peptides as anabaenopeptins in the samples was done. Finally, the MS protocol and identification tools suggested were applied to the analysis of characteristic water blooms from Spanish reservoirs.