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

Abstract

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.

Keywords: Anatoxin-a; Cyanobacteria; Cyanotoxin; High-resolution mass spectrometry; LC–HRMS; Non-target analysis.

Fig. 1

Structures, abbreviations and exact m/z ([M + H]⁺ or [M + 2H].²⁺, as indicated) of ATXs detected in field and culture samples in this study. An expanded version including isomer label assignments and retention time information is provided in the Supplementary information (Fig. S1)

Fig. 2

LC–HRMS extracted-ion chromatograms (m/z ± 3 ppm) of ATX (1), hATX (2), epoxyATX (18), cis- and trans-H₂ATX (5a, 5b), CYN, epiCYN, doCYN and phenylalanine (Phe) in RM-BGA

Fig. 3

LC–HRMS/MS analysis of a benthic-cyanobacterial-mat field sample in DDA scan mode showing the full-scan base-peak chromatogram with ATX (1), cis-H₂ATX (5a), trans-H₂ATX (5b) and Phe (A), and the extracted-ion chromatogram for m/z 166.1226 ± 5 ppm product ions from all MS/MS data, showing non-target detection of 3-OH-ATX (11b), H₃CO-ATX (13a, 13b) and CH₃SH-ATX (17) (B). The product-ion spectra of 11b (C), 13a (D), 13b (E), and 17 (F) from the corresponding DDA scans are shown in panes C–F, with the insets indicating the full-scan accurate mass, corresponding formula, and observed mass error for each precursor-ion

Fig. 4

Extracted-ion LC–HRMS chromatograms (± 5 ppm) showing additional ATX analogues detected in LC–HRMS analysis of a benthic-cyanobacterial-mat sample including ATX (1), hATX (2), H₂ATX (5), H₂hATX (6a, 6b), GSH-ATX (7a), γ-Glu-Cys-ATX (9b), 3-OH-ATX (11b), CH₃O-ATX (13a, 13b, 13c), CH₃S-ATX (17), 10-OH-H₂ATX (20a, 20b, 20c), 10-OH-H₂hATX (21a, 21b, 21c), 10-OH-ATX (22a, 22b) and 10-OH-hATX (23a)

Scheme 1

Treatment of an ATX or hATX standard with a conjugating nucleophile under basic conditions to promote Michael addition

Fig. 5

LC–HRMS/MS analysis of GSH-ATX (7b) in a benthic-cyanobacterial-mat sample (A, C) and isomer 7a and 7b in a semisynthetic preparation (B, D) showing extracted-ion chromatograms (exact m/z ± 5 ppm) (A, B) and product-ion spectra (C, D) of 7b at a CE of 10 eV

Scheme 2

Reduction of ATX and hATX with sodium borohydride

Scheme 3

Selective reduction of an H₂ATX standard using sodium cyanoborohydride

Fig. 6

LC–HRMS/MS comparison between natural (A, C) and semisynthetic (B, D) 10-OH-H₂ATX (20a, 20b, 20c) resulting from treatment of an H₂ATX standard with sodium cyanoborohydride, showing extracted-ion full-scan chromatograms (A, B) and product-ion spectra (C, D) of 20c

Fig. 7

LC–HRMS/MS analysis of isomers of H₂ATX (5a, 5b) and 10-OH-ATXOH (22a, 22b) in a benthic-cyanobacterial-mat field sample (A, E), a commercial H₂ATX standard (B, F), a laboratory culture C. issatschenkoi (C, G) and the reaction between ATX and sodium borohydride (D, H) showing extracted-ion chromatograms (m/z 168.1383 ± 5 ppm) (A–D) and CID spectra of the m/z 168.1383 peak at 7.5–7.6 min in each (E–H)

Fig. 8

Extracted-ion chromatograms from LC–HRMS analysis of preparations of C. issatschenkoi with acidic MeOH extraction: before (A) or after (B) freeze–thaw cell lysis, showing ATX (1) in both samples and carboxyATX (3) in the sample extracted prior to cell lysis, as well as cis-H₂ATX (5a), trans-H₂ATX (5b) and at least two isomers of 10-OH-H₂ATX (20a, 20b) in the sample extracted after cell lysis. All traces are shown on the same relative scale