Detection and Characterization of Cyanometabolite Complexes between Microcystin-RR and a Microguanidine by UHPLC-ESI-MS

Microcystins (MCs) are hepatotoxic cyclic peptides produced by cyanobacteria, with MC-RR being one of the most polar and commonly detected MC congeners in water collected during cyanobacterial harmful algal blooms (cHABs). Microguanidines (MGDs) are sulfated metabolites produced by Microcystis sp. that have not been reported during Lake Erie cHABs. Herein, both MGD AL772 (also abbreviated here as MGD) and previously unreported complex ions containing MC-RR and MGD AL772 were discovered after UHPLC-ESI-HRMS, MS/MS, and MS/MS/MS analyses of the Lake Erie cHAB samples in positive and negative modes. Initially, a triply charged [(MC-RR)₂-MGD+3H]³⁺ ion was detected at the same retention time as coeluting [MC-RR+2H]²⁺ and [MGD-H]⁻ ions. Characterization of this complex ion by HCD, CID, ETD and thiol derivatization indicates that two [MC-RR+2H]²⁺ ions are bonded to one [MGD-H]⁻, probably through interactions of positively charged guanidinium groups of arginine (R) residues and negatively charged MGD’s sulfate groups, to form the triply charged complex during ESI. Four other complexes, including [MC-RR-MGD] and its in-source fragment [(MC-RR-MGD)-SO₃], were also detected as positive and negative ions at the same time due to coelution of MC-RR and MGD. These findings not only underscore the importance of an efficient separation for quantification of cyanobacterial metabolites but also provide novel insights into their interactions during ESI, which enabled the present report on the complexes between MC-RR and a sulfated cyanobacterial metabolite, MGD AL772.