Nodularin nod

Fifteen cyanobacterial metabolites were analyzed as part of this study, including six hepatotoxins, six bioactive cyanopeptides, and three neurotoxins. For the hepatotoxins, certified reference material for microcystin-LR (MCLR) was purchased from the National Research Council of Canada Biotoxins program (Halifax, NS, Canada). Nodularin (NOD) (purity >94%), MCLA (>95%), MCYR (>90%), and MCRR (>90%) were purchased from Sigma-Aldrich (Milwaukee, WI, USA), and cylindrospermopsin (CYL) (>95%) was purchased from Abraxis (Warminster, PA, USA). For the peptides, anabaenopeptin B (Apt B) (>95%) and AptF (>95%), cyanopeptolin 1007 (Cpt1007) (>95%), Cpt 1021 (>95%), and Cpt 1040 (>95%), and microginin 690 (Mgn690) (>95%) were purchased from MARBIONC (Wilmington, NC, USA). Additionally, the neurotoxins anatoxin-a (ATX), homoanatoxin-a (hATX), and saxitoxin (SXT) were also targeted in this study. ATX fumarate (96%) was purchased from Tocris Bioscience (Minneapolis, MN, USA) as a racemic mixture, hATX (>95%) was purchased from Abraxis, and SXT was purchased from the National Research Council of Canada Biotoxins program (Halifax, NS, Canada).

All the extracts were later analyzed by HPLC Agilent 1290 Infinity II coupled to a hybrid mass spectrophotometer Agilent Q-TOF 6550, with an ionization source JetStream electrospray + i-Funnel. Samples were analyzed for eight MC variants ([Dhb⁷]-MC-LR, MC-RR, MC-YR, MC-LR, MC-LW, MC-LF, anatoxin-a (ANA) and nodularin (NOD) from SIGMA-ALDRICH). Compounds were separated in an Agilent Eclipse XDB-C18 4.6 × 150 mm, 5 mm column by Millipore water with 0.1 % formic acid (v/v, eluent A) and acetonitrile with 0.1% formic acid (v/v, eluent B). The elution program was 0–2 min 30% B, 6–12 min 90% B, with a linear increase of B between 2 and 6 min, and a 5-min post run at 30% B. The injection volume, flow and column temperature were 20 mL, 0.5 mL/min and 40 °C, respectively. MS operated in the positive mode and nitrogen was used as the drying and collision gas. The quadrupole was operated in the unit mode and four spectra/sec were recorded. Samples were quantified against a calibration curve and subsequently corrected for recovery (Table 7). Two replicas were obtained per sample and each replica was injected once.
The presence of phenylalanine was discarded because its theoretical m/z was 166.0863 and the employed method could separate it easily from anatoxin [58 (link)].