Based on current structural and statistical calculations, thousands of microcystins (MCs) can exist; yet, to date, only 246 MCs have been identified and only 12 commercial MC standards are available. Standard mass spectrometry workflows for known and unknown MCs need to be developed and validated for basic and applied harmful algal bloom research to advance. Our investigation focuses on samples taken in the spring of 2018 from an impoundment fed by Oser and Bischoff Reservoirs, Indiana, United States of America (USA). The dominant cyanobacterium found during sampling was Planktothrix agardhii
. The goal of our study was to identify and quantify the MCs in the impoundment sample using chemical derivatization and mass spectrometry. Modifying these techniques to use online concentration liquid chromatography tandem mass spectrometry (LC–MS/MS), two untargeted MCs have been identified, [d
]-MC-LR and tentative [Dhb7
]-MC-YR is not yet reported in the literature to date, and this was the first reported incidence of Dhb MCs in the United States. Furthermore, it was discovered that the commercially available [d
]-MC-RR standard was [d
]-MC-RR. This study highlights a workflow utilizing online concentration LC–MS/MS, high-resolution MS (HRMS), and chemical derivatization to identify isobaric MCs.
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