Mc lw

All chemicals were reagent grade or higher in quality. Formic acid (FA) and ammonium hydroxide solution (NH₄OH) were purchased from Sigma-Aldrich (Milan, Italy). HPLC gradient grade methanol (MeOH), acetonitrile (ACN) and ultrapure water (H₂O) were supplied by Carlo Erba (Cornaredo, Italy). Strata™-X cartridges (200 mg, 3 mL) were purchased from Phenomenex (Castel Maggiore, Italy). DA (≥90%), OA sodium salt (MQ 100) and analytical grade standard MC-RR, MC-LR, MC-YR and MC-LW solutions were supplied by Sigma-Aldrich (Milan, Italy). Individual stock standard solutions of all analytes (1 μg mL⁻¹) were prepared in MeOH, stored in the dark (−20 °C) and renewed weekly.

MCLR, MCLF, MCLA, MCLY, and MCLW were purchased from Sigma (Saint-Quentin Fallavier, France). PP2A was purchased from New England Biolabs Inc (Beverly, MA, USA). Bovine serum albumin, dithiothreitol, MnCl₂, P-nitrobenzene disodium orthophosphate, sodium thiosulfate, tris(hydroxymethyl) aminomethane, and other reagents were purchased from Sinopharm (Shanghai, China).

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)].