Acetonitrile

Brain tissue homogenates, including frontal cortex, dorsal and ventral hippocampus, were used to measure levels of glutamate (Glu), glutamine (GLN), and Gamma Aminobutyric Acid (GABA). A YL9112 Plus Isocratic HPLC Pump (YOUNG IN Chromass Inc., Korea) coupled with a DECADETM Elite Electrochemical Detector (Antec@Scientific, USA) was used. HypersilTM ODS C18, 250 mm × 10 mm × 5 μm column (Thermo Fisher ScientificTM, Massachusetts, USA) was used with pre-column derivatization, as previously described, with some minor modifications [19 (link)]. In brief, the voltage of the working electrode was set at +800 mV, and the mobile phase consisted of acetonitrile (Chem-Lab, Belgium): 100 mM monosodium-phosphate buffer pH 5.5, containing 0.5 mM Na2EDTA and 0.1 M Citric Acid 1-hydrate (PanReac AppliChem, Germany). Samples were initially diluted 1:5 with ddH₂O, then further diluted with 0.1 M Borax buffer (Sigma-Aldrich, St. Louis, USA), including o-Phthalaldehyde (Sigma-Aldrich), pH 10.4. External standards were used each day to generate the calibration curve. Quantification of Glu, GLN, and GABA was performed by comparison of the area under the curve with that of reference external standards using Clarity Software (Data-Apex, Czech Republic), and cycling rates (GLN/Glu and GLN/GABA) were calculated.

Htyr (≥98%) was purchased from Extrasynthèse (Genay, France) and Tyr (≥98%) from Alfa Aesar GmdH & Co KG, (Karlsruhe, Germany), CA (98%) and DHPA (98%) were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Analytical-grade methanol, HPLC grade methanol, acetonitrile and water were obtained from ChemLab (Zeldegem, Belgium). Phosphoric acid (≥85%) and sulphuric acid (95–98%) were supplied by Sigma-Aldrich (Steinheim, Germany). Sodium carbonate anhydrous and Folin-Ciocalteu (F-C) reagent were obtained from ChemLab (Zeldegem, Belgium). Polyvinylidene fluoride (PVDF) membrane filters (0.22 µm) were from Schleicher & Schuell, (Dassel, Germany).

Chromatographic quality organic solvents, namely acetonitrile and methanol were obtained from Chem-Lab (Zedelgem, Belgium). Chloroform, carbon tetrachloride, 1-undecanol and 1-octanol were purchased from Sigma Aldrich (St. Louis, MO, USA). The ILs 1-hexyl-3-methylimidazolium hexafluorophosphate (C₁₀H₁₉F₆N₂P) [HMIM][PF6], lithium-imide bis(trifluoromethylsulfonyl)imide (LiC₂F₆NO₄S₂) [LiTFSI] and 1-hexyl-3-methylimidazolium chloride (C₁₀H₁₉ClN₂) [HMIM Cl], were supplied by IOLITEC (Heilbronn, Germany). Other reagents, including formic acid and sodium chloride, were acquired from Sigma. Anhydrous magnesium sulfate from Análisis Vínicos (Tomelloso, Spain), hydrochloric acid from Riedel-de-Häen (Wunstarfer, Germany) and phosphoric acid from Panreac Química S.A (Barcelona, Spain) were also used. Water was previously purified using a Milli-Q system (Millipore, Bedford, MA, USA).
The cyanotoxin and phycotoxin standards were obtained from Cifga S.A. (Lugo, Spain). The standard solutions of cyanotoxins—microcystine LR (MC-LR) and RR (MC-RR) and nodularin (NOD)—were supplied at 10 µg g⁻¹, while okadaic acid (OA) was supplied at 16 µg g⁻¹ and domoic acid (DA) at 40 µg g⁻¹. All were stored in the dark at −20 °C, and standard working solutions were prepared daily diluting the toxins with ultrapure water.

For HPLC analysis methanol (HPLC, 99.9%) and hexane (HPLC 95%) were purchased from Merck (Darmstadt, Germany). 2-Propanol (Chromasolv) was from Riedel de Haën (Seelze, Germany). Acetonitrile (HPLC, 99.9%), acetone (HPLC, 99.8%), and water (HPLC grade) were obtained from ChemLab (Zeldegem, Belgium). Phosphoric acid (≥85%) and sulphuric acid (95–98%) were supplied by Sigma-Aldrich (Steinheim, Germany). Sodium carbonate (anhydrous) and Folin-Ciocalteu (F-C) reagent were obtained from ChemLab. Other solvents and reagents were of the appropriate grade from various suppliers. Htyr (≥98%) was purchased from Extrasynthèse (Genay, France) and Tyr (≥98%) from AlfaAesar GmdH & Co KG, (Karlsruhe, Germany). Caffeic acid (CA) (98%) was from Sigma–Aldrich and α-Τ (>96%) were from Fluka Chemie GmbH (Buchs, Switzerland). Polyvinylidene fluoride (PVDF) membrane filters (0.22 µm) and cellulose acetate membrane filters (0.45 µm) were from Schleicher & Schuell, (Dassel, Germany).

Caffeic acid (3,4-Dihydroxycinnamic acid) was acquired from Sigma-Aldrich, St. Louis, MO, and used as received without further purification. The molecular structure of caffeic acid in non-hydrolyzed form is illustrated in Table 1. Iron (II) sulfate heptahydrate (FeSO₄•7H₂O) was acquired from Panreac, Barcelona, Spain, and hydrogen peroxide (H₂O₂ 30% w/w) and titanium (IV) oxysulfate solution 1.9–2.1%, for determination of hydrogen peroxide, were acquired from Sigma-Aldrich, St. Louis, MO, USA. Sodium sulfite anhydrous (Na₂SO₃) was acquired from Merk, Darmstadt, Germany. Trifluoroacetic acid (HPLC grade, ≥99.0%) was acquired from Riedel-de Haën, Seelze, Germany, and acetonitrile (HPLC grade, ≥99.9%) was acquired from Chem-Lab, Zedelgem, Belgium. For pH adjustment, sodium hydroxide (NaOH) from Labkem, Barcelona, Spain, it was used, along with sulfuric acid (H₂SO₄, 95%) from Scharlau, Barcelona, Spain. Deionized water was used to prepare the respective solutions.