Analysis of total microcystins and nodularins by oxidative cleavage of their ADMAdda, DMAdda, and Adda moieties

Amanda J Foss; Christopher O Miles; Alistair L Wilkins; Frode Rise; Kristian W Trovik; Kamil Cieslik; Mark T Aubel

doi:10.1016/j.acax.2020.100060

Analysis of total microcystins and nodularins by oxidative cleavage of their ADMAdda, DMAdda, and Adda moieties

Anal Chim Acta X. 2020 Sep 2:6:100060. doi: 10.1016/j.acax.2020.100060. eCollection 2020 Nov.

Authors

Amanda J Foss¹, Christopher O Miles^{2

3}, Alistair L Wilkins^{3

4

5}, Frode Rise⁵, Kristian W Trovik⁵, Kamil Cieslik¹, Mark T Aubel¹

Affiliations

¹ GreenWater Laboratories/CyanoLab, 205 Zeagler Drive, Palatka, FL, 32177, USA.
² Measurement Science and Standards, National Research Council, 1411 Oxford Street, Halifax, NS, B3H 3Z1, Canada.
³ Norwegian Veterinary Institute, P. O. Box 750, Sentrum, N-0106, Oslo, Norway.
⁴ Chemistry Department, University of Waikato, Private Bag 3105, 3240, Hamilton, New Zealand.
⁵ Department of Chemistry, University of Oslo, P.O. Box 1033, N-0315, Oslo, Norway.

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.

Keywords: ADMAdda; ADMAdda, 3S-amino-9S-acetyloxy-2S,6,8S-trimethyl-10-phenyldeca-4E,6E-dienoic acid; Adda; Adda, 3S-amino-9S-methoxy-2S,6,8S-trimethyl-10-phenyldeca-4E,6E-dienoic acid; DMAdda; DMAdda, 3S-amino-9S-hydroxy-2S,6,8S-trimethyl-10-phenyldeca-4E,6E-dienoic acid; MAPB, 2R-methyl-3S-acetyloxy-4-phenylbutanoic acid; MHPB, 2R-methyl-3S-hydroxy-4-phenylbutanoic acid; MMPB; MMPB, 2R-methyl-3S-methoxy-4-phenylbutanoic acid; MOMAPH, 2-methyl-3-oxo-4R-methyl-5S-acetyloxy-6-phenylhexanoic acid; MOMHPH, 2-methyl-3-oxo-4R-methyl-5S-hydroxy-6-phenylhexanoic acid; MOMMPH, 2-methyl-3-oxo-4R-methyl-5S-methoxy-6-phenylhexanoic acid; Microcystin; Microcystin, MC; NOD, Nodularin; Nodularin.