Methanol

Microcystin standards (MC-LR, MC-RR and MC-YR) with purities ≥95% were purchased from Alexis Biochemicals (Lausen, Switzerland). The stock solution of three MC congeners was prepared in 1 mL of Methanol solution at a concentration of 100 mg/L for each variant, which was kept at −20 °C for later use. The C18 solid phase extraction (SPE) cartridges (500 mg, 6 mL) used in MCs analysis were obtained from Anpel Company (Shanghai, China). The standard compounds GEO, MIB and 2-isobutyl-3-methoxypyrazine (IBMP, as the internal standard) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) with the concentration of 100 mg/L in Methanol, while CYC and ION with purities ≥97% were purchased from Adamas Reagent Co., Ltd. (Basel, Switzerland). The stock solution of 1 mg/L with four target T&O compounds prepared in Methanol was stored in the dark at 4 °C. Sodium chloride (NaCl, Sigma, St. Louis, MO, USA) was added to the samples to enhance the T&O compounds extraction from water.
Methanol, trifluoracetic acid, acetic acid, acetonitrile and acetone were of HPLC grade from Tedia Company (Fairfield, OH, USA). Water used throughout the work was from a Milli-Q water purification system (Millipore, Billerica, MA, USA). All other reagents used were of analytical grade or better.

All reagents, specifically 2,2´-diphenyl-1-picrylhydrazyl (DPPH), 2,2´-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), were purchased from Sigma–Aldrich (St. Louis, MO, USA). HPLC-grade phenolic standards (protocatechuic acid, (+)-catechin, p-hydroxybenzoic acid, chlorogenic acid, (−)-epicatechin, caffeic acid, p-coumaric acid, ferulic acid, rutin, quercetin, quercetin-3-rhamnoside, cAMP, and cGMP) were also purchased from Sigma–Aldrich (St. Louis, MO, USA). HPLC grade sugar standards (glucose, fructose, and sucrose), ascorbic acid (AA), phloridzin and kaempferol were purchased from Yuanye Biotechnology Ltd. (Shanghai, China). Methanol, acetonitrile and formic acid were purchased from Tedia Company, Inc. (Fairfield, CT, USA). Folin-Ciocalteu reagent and all other chemicals were obtained from Sinopharm Chemical Reagent Co. (Shanghai, China).

EE was solubilized in methanol (Tedia) (1 mg/mL), and 10 μL of this sample was analyzed using LaChrom Elite HPLC system (Hitachi, Tokyo, Japan) liquid chromatograph equipped with L2130 pump, auto-sampler L2200, L2300 column oven was set at 25 °C and an L2455 diode array detector (DAD) (Hitachi, Tokyo, Japan). The C-18 column (5 μm, 150 mm × 4.6 mm) was used in combination with an appropriate guard column (4.0 mm × 4.0 mm; 5 μm of particle size) (Merck, Darmstadt, Germany). The analysis was performed at a wavelength fixed at 354 nm. The eluents used were aqueous phosphoric acid (Merck, Darmstadt, Germany) (1%) (solvent A) and acetonitrile (J. T. Baker, Leicestershire, UK) (solvent B). The gradient employed was 90% A and 10% B for 0 min, 70% A and 30% B for 40 min, 50% A and 50% B for 50 min, 90% A and 10% B for 51 min, and 90% A and 10% B for 55 min at a flow rate of 0.6 mL/min. Data acquisition was performed using ExChrom Elite software (version 3.3.2 SP1) (Scientific Software Inc., Santa Clara, CA, USA). The compounds present in the extract were compared according to their UV–Vis spectra (similarity index > 0.99) and retention times with commercial standards, as previously described [44 ].