Mc yr

MC-LR, MC-RR, and MC-LA were purchased from Cayman Chemical (Ann Arbor, MI, USA). MC-YR, MC-LF, and MC-LW were purchased from Enzo Life Sciences (Farmingdale, NY, USA). C₂D₅ MC-LR was purchased from Cambridge Isotope Laboratories (Tewksbury, MA, USA). MC-LR-Cys was synthesized by the Westrick group (Wayne State University, Detroit, MI, USA) [46 (link)]. ACS-grade zinc sulfate (ZnSO₄) and HPLC-grade water, methanol (CH₃OH), and acetonitrile (CH₃CN) were purchased from Fisher Scientific (Pittsburgh, PA, USA). Reagent-grade formic acid (FA) was purchased from Sigma (St. Louis, MO, USA).

MCs (MC-LR, MC-YR, MC-RR, MC-LA, MC-LY and MC-LF) and NOD standards were purchased from Enzo Life Sciences, Ltd. (UK), CYN was obtained from n’Tox (France) and ATX-A was purchased from the National Research Council, Canada. MMPB was purchased from Wako Pure Chemical Industries, Ltd. (Japan). CYN N¹⁵-labelled internal standard (N¹⁴ → N¹⁵) was a gift from the Federal Institute for Materials Research and Testing (BAM), Berlin, Germany. Acetonitrile and methanol, LC-MS Chromasolv grade, dichloromethane, formic acid, trifluoroacetic acid, potassium permanganate, sodium (meta) periodate and sodium bisulfite were all purchased from Sigma-Aldrich, UK, as were the ENVI-Carb solid-phase extraction (SPE) cartridges (250 mg, 3 cm³). Oasis HLB and PRiME SPE cartridges (60 mg, 3 cm³) were purchased from Waters, Ireland. The water used was supplied from an in-house Milli-Q water system (Millipore, Ltd., UK), with conductivity and total organic content (TOC) of the water being 18 MΩ and 3 ppb, respectively.

The solvents for the extraction and for the basis of the mobile phase were UHPLC-MS grade. All toxin standards for MCs (MC-LR, MC-RR, MC-LA, MC-LY, MC-LF, MC-LW, MC-YR, MC-WR) and NOD came from Enzo Life Sciences^® (Antwerp, Belgium) and were received under the form of a solid powder. After dilution in 100% methanol (MeOH), mixed stock solutions were prepared in 50% methanol (MeOH) (50% Milli-Q water with 1% acetic acid (v/v)). The stock and the intermediate solutions were stored at −20 °C.

ANA-a, CYN, MC-LR, [Asp³] MC-LR and MC-RR (purity ≥ 99%) were purchased from the National Research Council of Canada (Halifax, NS, Canada). Nodularin-R (NOD-R), [Asp³] MC-RR, MC-YR, LA, LY, LW, LF, WR, HtyR and HilR (purity ≥ 95%) were purchased from Enzo Life Science (Farmingdale, NY, USA). Homoanatoxin-a (HANA-a, purity ≥ 99%) was obtained from Abraxis, Inc. (Warminster, PA, USA), Anabaenopeptin A and B (AP-A, AP-B, purity ≥ 90%) from Cyano Biotech GmbH (Berlin, Germany), and ¹⁵N₁₀-MC-LR (95%) from Cambridge Isotopes Laboratories, Inc. (Tewksbury, MA, USA). Individual stock solutions of ANA-a, CYN, MC-LR, [Asp³] MC-LR and MC-RR were kept at −20 °C for a maximum of six months. All other individual stock solutions were prepared in methanol (MeOH) at a concentration of 25 mg L⁻¹ and were kept at −20 °C for a maximum of one year. Primary working solutions were prepared at a concentration of 100 µg L⁻¹ for targeted cyanotoxins and 9 µg L⁻¹ for internal standards (Iss: ¹⁵N₁₀-MC-LR and NOD-R) by dilution in MeOH of individual stock solution aliquots. Subsequent working solutions were prepared daily by dilution in water to give solutions of the desired concentration. All organic solvents and water used for dilutions were of HPLC grade purity from Fisher Scientific (Whitby, ON, Canada).

Three congeners of MCs (MC-LR, MC-RR, and MC-YR) (99% purity) and Cylindrospermopsin standard (95% purity) were purchased from Enzo Life Sciences (Lausen, Switzerland). Deionized water (18.2 MΩ cm resistivity) was obtained from a Milli-Q water purification system (Millipore, Bedford, MA, USA). HPLC-grade methanol, dichloromethane (DCM), formic acid (FA), acetonitrile, and sodium hydroxide (NaOH) were supplied by Merck (Darmstadt, Germany). For the SPE, C18 cartridges were Bakerbond^® (500 mg, 6 mL), purchased from Dicsa (Andalucía, España), and graphitized carbon cartridges were BOND ELUT^® (500 mg, 6 mL), supplied by Agilent Technologies (Amstelveen, The Netherlands). For UHPLC–MS/MS analyses, reagents were of LC–MS grade: Water and acetonitrile were supplied by VWR International (Fontenay-sous-Bois, France) and formic acid by Fluka (Steinheim, Germany). A standard multitoxin solution containing 100 µg L⁻¹ of each cyanotoxin (MC-LR, MC-RR, MC-YR, and CYN) was prepared in 20% MeOH to be further diluted to three different concentrations (5, 20, and 50 µg L⁻¹), used as working solutions. Lettuce control samples (without toxins) were obtained from a local supermarket, ready for human consumption.

LC-MS grade acetonitrile, water and formic acid and HPLC-grade methanol and water were purchased from Fisher (ThermoFisher, UK). Reference toxin standards of microcystins (MC-LR, MC-RR, MC-YR, MC-WR, MC-LW, MC-LA, MC-LY, MC-LF, MC-HtyR, MC-HiLR, [Asp3]-MC-LR/[Dha7]-MC-LR) and NOD ≥95% were as per Enzo Life Sciences (Exeter, UK) (Fig. S1). A certified standard of [Dha7]-MC-LR and a pre-certified freeze-dried matrix reference material of blue-green algae (RM-BGA, Lot 201301) containing a range of MCs were purchased from the Institute of Biotoxin Metrology, National Research Council Canada (Ontario, Canada).
A mixed stock solution was prepared by combining aliquots of each toxin to give a final concentration of 327 μg/L. For external calibration, a seven point calibration curve was prepared by serial dilution with methanol/water (1:1, v/v) in the range of 0.33–327 μg/L for each toxin and stored at – 18 °C. A quality control reference material (RM-BGA, National Research Council, Halifax, Canada) was prepared, with toxins extracted in the supernatant after 28 mL of methanol/water (1:1, v/v) + 0.1% acetic acid were added to RM-BGA (280 mg) and subsequent centrifugation (4500  $\times$ g; 10 min).
Shellfish diet 1800 (approximately 7.4 × 10¹¹ cells/mL) was purchased from ReedMariculture Inc., (US) and dilutions were made in water/seawater (10:0.86, v/v).