Mc la

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.

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).

CYN, ANA, MC-LR, MC-RR, MC-YR, MC-LY, MC-LW, and MC-LF (99%) standards were purchased from Cyano Biotech GmbH (Berlin, Germany). NOD and MC-LA were purchased from ENZO life Science (Lausen, Switzerland). Methanol (MeOH), acetonitrile (ACN), and water HPLC grade were obtained from Fisher Scientific (Leics, UK). FA (98%) was purchased from Fluka (Steinheim, Germany). NH₄OH (25%) was obtained from Sigma-Aldrich (Steinheim, Germany). Sodium chloride, potassium chloride (99.5%), and sodium carbonate (99.9%) were purchased from Merck (Darmstadt, Germany). Calcium chloride (93%), magnesium chloride (98%), and HEPES (99.5%) were obtained from Sigma-Aldrich (Steinheim, Germany). AFW was prepared according to Lipschitz et al. [44 (link)], and Na₂CO₃ 1M was used for AFW pH adjustment

The extracts were separated using a Purospher STAR RP-18 end-capped column (30 × 4 mm, 3 μm particle size, Merck, Darmstadt, Germany) at 30 °C as described by Spoof et al. [62 (link)]. The mobile phase consisted of 0.5% formic acid (A) and acetonitrile with 0.5% formic acid (B) at a flow rate of 0.5 mL/min with the following gradient program: 0 min 25% B, 10 min 70% B, 11 min 70% B. The injection volume was 10 μL. Identification and quantification of the MCs ([Asp3]-MC-RR, MC-RR, MC-YR, [Asp3]-MC-LR, MC-LR, MC-LW, MC-LF, MC-LA, standards purchased at Enzo Life Sciences, Lörrach, Germany) was performed in the MRM (Multiple Reaction Monitoring) mode with the transitions given by Fastner et al. [63 (link)].

Instrument solvents used for preparation of mobile phases were of LC-MS-grade (Fisher Optima, ThermoFisher, London, UK) and all chemicals were LC-MS reagent grade where possible, otherwise HPLC grade. Sample preparation reagents were all HPLC grade. Cyanotoxin reference toxin standards comprising the microcystin analogues (MC-RR, MC-LA, MC-LY, MC-LF, MC-LW, MC-YR, MC-WR, MC-HilR, MC-HtyR, MC-LR, [Asp3] MC-LR), Nodularin (Nod), ATX, Cylindrospermopsin (CYN) were all obtained from Enzo Life Sciences (Enzo, Exeter, UK), with secondary calibration check standards and Saxitoxin (STX) standard prepared using certified reference solutions from Biotoxin Metrology, National Research Council of Canada (NRCC, Halifax, Nova Scotia, Canada). Enzo reference standards received as solid powders were dissolved in suitable volumes of 50% aqueous methanol, to form stock solutions. A mixed stock solution was prepared by combining aliquots of each stock, followed by a seven-level suite of working calibration solvent standards resulting in a calibration range between 1.0 ng/mL to 500 ng/m per toxin. Phe was also obtained from Merck (Poole, UK) and used for confirmation of resolution from ATX, given the isobaric nature of the two compounds and the potential for false positive detection of ATX in matrices containing Phe [62 (link)].