Analysis of free and metabolized microcystins in samples following a bird mortality event

Foss, Amanda J.; Miles, Christopher O.; Samdal, Ingunn A.; Løvberg, Kjersti E.; Wilkins, Alistair L.; Rise, Frode; Jaabaek, J. Atle H.; McGowan, Peter C.; Aubel, Mark T.

doi:10.1016/j.hal.2018.10.006

Cited by 36 publications

(66 citation statements)

References 45 publications

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“…The [M + H] + ion for both sulfone-8, and sulfoxide-6 after neutral loss of CH3SOH, showed the expected characteristic series of microcystin product ions. Further examination of the LC-HRMS/MS chromatograms revealed the presence of a peak corresponding to [D-Leu 1 ]MC-MR (7) that was recently also reported by Foss et al [11] in a cyanobacterial bloom sample, together with 6 and 7. Microcystins 6-8 from this sample and from M. aeruginosa CPCC-464 showed identical retention times and mass spectral characteristics.…”

Section: Resultssupporting

confidence: 75%

“…LC-HRMS chromatograms with and without mercaptoethanol derivatization are shown in Figures S1 and S2. Compounds 1 and 3 were hypothesized to be [D-Leu 1 ]MC-LY and [D-Leu 1 ]MC-LR, respectively [5], while the MS characteristics of 6 are consistent with [D-Leu 1 ]MC-M(O)R, tentatively identified recently by LC-HRMS/MS in cyanobacterial blooms in Canada and the USA [9,11].…”

Section: Resultssupporting

confidence: 59%

“…The fact both 1 and 3 are biosynthesized together by the MC synthetase of M. aeruginosa strain CPCC-464, and that 1 was subsequently found to have similar inhibitory potency to MC-LR (2) against protein phosphatase 2A (PP2A) (Figure 4), both indicate that 1 has the same absolute stereochemistry as 2 and 3 and that 1 is therefore [D-Leu 1 ]MC-LY ( Figure 1). Authentic 3 in a cyanobacterial bloom extract from Poplar Island, MD, USA, whose structure has been verified as [D-Leu 1 ]MC-LR by purification and NMR analysis [11], had identical retention time and product ion spectra to the peak for 3 in CPCC-464 when analyzed by LC-HRMS/MS, thus verifying its identity as proposed by Hollingdale et al [5]. LC-HRMS/MS also showed the presence of a minor microcystin with [M + H] + at m/z 1071.5556 (C51H79O13N10S + , Δ 1.2 ppm) consistent with [D-Leu 1 ]MC-M(O)R (6) tentatively identified in bloom samples from south-western Ontario, Canada [9] and Maryland, USA [11], by untargeted LC-HRMS/MS methods and selective chemical oxidation.…”

Section: Resultsmentioning

confidence: 95%

“…Authentic 3 in a cyanobacterial bloom extract from Poplar Island, MD, USA, whose structure has been verified as [D-Leu 1 ]MC-LR by purification and NMR analysis [11], had identical retention time and product ion spectra to the peak for 3 in CPCC-464 when analyzed by LC-HRMS/MS, thus verifying its identity as proposed by Hollingdale et al [5]. LC-HRMS/MS also showed the presence of a minor microcystin with [M + H] + at m/z 1071.5556 (C 51 H 79 O 13 N 10 S + , ∆ 1.2 ppm) consistent with [D-Leu 1 ]MC-M(O)R (6) tentatively identified in bloom samples from south-western Ontario, Canada [9] and Maryland, USA [11], by untargeted LC-HRMS/MS methods and selective chemical oxidation. The identity of this compound in the culture was further verified by targeted LC-HRMS/MS, together with oxidation to its sulfone (8) using Oxone [22] (see Figures S20 and S21).…”

Section: Resultsmentioning

confidence: 95%

“…A careful non-targeted LC-MS analysis of a field sample by Foss et al [11] recently reported more than 20 Leu 1 -containing MCs in a cyanobacterial bloom, with [D-Leu 1 ]MC-LR (3) as the major component, but no 1 was detected. Including the present study, 1 now appears to have been detected in samples originating from three different locations in Canada [5,9], but so far, nowhere else in the world.…”

Section: Resultsmentioning

confidence: 99%

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Isolation and Characterization of [D-Leu1]microcystin-LY from Microcystis aeruginosa CPCC-464

LeBlanc

Merkley

Thomas

et al. 2020

Toxins

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[D-Leu1]MC-LY (1) ([M + H]+ m/z 1044.5673, Δ 2.0 ppm), a new microcystin, was isolated from Microcystis aeruginosa strain CPCC-464. The compound was characterized by 1H and 13C NMR spectroscopy, liquid chromatography–high resolution tandem mass spectrometry (LC–HRMS/MS) and UV spectroscopy. A calibration reference material was produced after quantitation by 1H NMR spectroscopy and LC with chemiluminescence nitrogen detection. The potency of 1 in a protein phosphatase 2A inhibition assay was essentially the same as for MC-LR (2). Related microcystins, [D-Leu1]MC-LR (3) ([M + H]+ m/z 1037.6041, Δ 1.0 ppm), [D-Leu1]MC-M(O)R (6) ([M + H]+ m/z 1071.5565, Δ 2.0 ppm) and [D-Leu1]MC-MR (7) ([M + H]+ m/z 1055.5617, Δ 2.2 ppm), were also identified in culture extracts, along with traces of [D-Leu1]MC-M(O2)R (8) ([M + H]+ m/z 1087.5510, Δ 1.6 ppm), by a combination of chemical derivatization and LC–HRMS/MS experiments. The relative abundances of 1, 3, 6, 7 and 8 in a freshly extracted culture in the positive ionization mode LC–HRMS were ca. 84, 100, 3.0, 11 and 0.05, respectively. These and other results indicate that [D-Leu1]-containing MCs may be more common in cyanobacterial blooms than is generally appreciated but are easily overlooked with standard targeted LC–MS/MS screening methods.

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Section: Resultssupporting

confidence: 75%

Section: Resultssupporting

confidence: 59%

Section: Resultsmentioning

confidence: 95%

Section: Resultsmentioning

confidence: 95%

Section: Resultsmentioning

confidence: 99%

See 3 more Smart Citations

Isolation and Characterization of [D-Leu1]microcystin-LY from Microcystis aeruginosa CPCC-464

LeBlanc

Merkley

Thomas

et al. 2020

Toxins

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Recent developments in the methods of quantitative analysis of microcystins

Kumar

Rautela

Kesari

et al. 2020

J Biochem & Molecular Tox

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Cyanotoxins are produced by the toxic cyanobacterial species present in algal blooms formed in water bodies due to nutrient over-enrichment by human influences and natural environmental conditions. Extensive studies are available on the most widely encountered cyanotoxins, microcystins (MCs) in fresh and brackish water bodies. MC contaminated water poses severe risks to human health, environmental sustainability, and aquatic life. Therefore, commonly occurring MCs should be monitored. Occasionally, detection and quantification of these toxins are difficult due to the unavailability of pure standards. Enzymatic, immunological assays, and analytical techniques like protein phosphatase inhibition assay, enzymelinked immunosorbent assay, high-performance liquid chromatography, liquid chromatography-mass spectrometry, and biosensors are used for their detection and quantification. There is no single method for the detection of all the different types of MCs; therefore, various techniques are often combined to yield reliable results. Biosensor development offered a problem-solving approach in the detection of MCs due to their high accuracy, sensitivity, rapid response, and portability. In this review, an endeavor has been made to uncover emerging techniques used for the detection and quantification of the MCs.

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Liquid chromatography–high-resolution tandem mass spectrometry of anatoxins, including new conjugates and reduction products

et al. 2023

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Anatoxins (ATXs) are a potent class of cyanobacterial neurotoxins for which only a handful of structural analogues have been well characterized. Here, we report the development of an LC–HRMS/MS method for the comprehensive detection of ATXs. Application of this method to samples of benthic cyanobacterial mats and laboratory cultures showed detection of several new ATXs. Many of these result from nucleophilic addition to the olefinic bond of the α,β-unsaturated ketone functional group of anatoxin-a (ATX) and homoanatoxin-a (hATX), analogous to the conjugation chemistry of microcystins, which contain similar α,β-unsaturated amide functionality. Conjugates with glutathione, γ-glutamylcysteine, methanethiol, ammonia, methanol and water were detected, as well as putative C-10 alcohol derivatives. Structural confirmation was obtained by simple and selective analytical-scale semisynthetic reactions starting from available ATX standards. Methanol, water and ammonia conjugates were found to result primarily from sample preparation. Reduction products were found to result from enzymatic reactions occurring primarily after cell lysis in laboratory cultures of Kamptonema formosum and Cuspidothrix issatschenkoi. The relative contributions of the identified analogues to the anatoxin profiles in a set of 22 benthic-cyanobacterial-mat field samples were estimated, showing conjugates to account for up to 15% of total ATX peak area and 10-hydroxyanatoxins up to 38%. The developed methodology, new analogues and insight into the chemical and enzymatic reactivity of ATXs will enable a more comprehensive study of the class than possible previously.

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Analysis of free and metabolized microcystins in samples following a bird mortality event

Cited by 36 publications

References 45 publications

Isolation and Characterization of [D-Leu1]microcystin-LY from Microcystis aeruginosa CPCC-464

Isolation and Characterization of [D-Leu1]microcystin-LY from Microcystis aeruginosa CPCC-464

Recent developments in the methods of quantitative analysis of microcystins

Liquid chromatography–high-resolution tandem mass spectrometry of anatoxins, including new conjugates and reduction products

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