Glacial acetic acid acoh

The chemicals and reagents used were of analytical or higher grade. Acetonitrile (MeCN, LC-MS grade) and methanol (MeOH, HPLC grade) were purchased from Scharlab (Barcelona, Spain) and VWR (Barcelona, Spain), respectively. Ultrapure water was obtained from a Milli-Q Gradient system fed with an Elix Advantage-10 (Millipore Iberica, Madrid, Spain). Ammonium hydroxide (NH₄OH, 25%), sodium hydroxide (NaOH > 99%), formic acid (98–100%), and glacial acetic acid (AcOH) were obtained from Merck (Barcelona, Spain), and hydrochloric acid (HCl, 37%) was obtained from Panreac (Barcelona, Spain). The graphitized carbon Supelclean ENVICarb (250 mg/3 mL) cartridges were acquired from Supelco (Bellefonte, PA, USA).
Certified reference standard (CRM) for GYM A was acquired from the Institute for Marine Biosciences, National Research Council (NRC) (Halifax, NC, Canada) and for TTX from Cifga S.A. (Lugo, Spain). Working standard solutions were prepared by diluting the stock solution with 50% methanol for GYM A or acetic acid 0.1 mM for TTX.

Frozen C. intybus leftovers were kindly provided by BioSphere (Bertinoro, Italy).
EtOH (ACS grade, ≥99%) (Sigma–Aldrich, Milan, Italy) and methanol (MeOH, HPLC, ≥99.9%) were used for extractions, total phenolic micro-assays and SPE purifications. Acetonitrile for HPLC analyses (MeCN, HPLC Plus, ≥99.9%) was purchased from Sigma–Aldrich, while Milli-Q H₂O was obtained in the laboratory from a Milli-Q Reference A + System (Merck Millipore, Darmstadt, DE, USA). Glacial acetic acid (AcOH, ≥96%) was purchased from Merck (Darmstadt, DE, USA). Standards of gallic, p-hydroxybenzoic, caffeic, ellagic, p-coumaric, ferulic, chlorogenic, vanillic, syringic, protocatechuic and 3,4-dimethoxybenzoic (veratric) acids, (−)-epicatechin, apigenin-3-glucoside, luteolin-3-glucoside as well as the Folin–Ciocalteau reagent and sodium carbonate for total phenolic assays were purchased from Sigma-Aldrich (Milan, Italy).