| Abstract | Microcystins (MCs) and nodularins (NODs) exhibit high structural variability, including modifications of the Adda (3S-amino-9S-methoxy-2S,6,8S-trimethyl-10-phenyldeca-4E,6E-dienoic acid) moiety. Variations include 9-O-desmethylAdda (DMAdda) and 9-O-acetylDMAdda (ADMAdda) which, unless targeted, may go undetected. Therefore, reference standards were prepared of [ADMAdda⁵]MCs and [DMAdda⁵]MCs, which were analyzed using multiple approaches. The cross-reactivities of the [DMAdda⁵]- and [ADMAdda⁵]MC standards were similar to that of MC-LR when analyzed with a protein phosphatase 2A (PP2A) inhibition assay, but were <0.25% when analyzed with an Adda enzyme-linked immunosorbent assay (ELISA). Oxidative cleavage experiments identified compounds that could be used in the analysis of total MCs/NODs in a similar fashion to the 2R-methyl-3S-methoxy-4-phenylbutanoic acid (MMPB) technique. Products from oxidative cleavage of both the 4,5- and 6,7-ene of Adda, DMAdda and ADMAdda were observed, and three oxidation products, one from each Adda variant, were chosen for analysis and applied to three field samples and a Nostoc culture. Results from the oxidative cleavage method for total Adda, DMAdda, and ADMAdda were similar to those from the Adda-ELISA, PP2A inhibition, and LC-MS/MS analyses, except for the Nostoc culture where the Adda-ELISA greatly underestimated microcystin levels. This oxidative cleavage method can be used for routine analysis of field samples and to assess the presence of the rarely reported, but toxic, DMAdda/ADMAdda-containing MCs and NODs. |
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