Millipore direct q3 uv system

Phosphatidylcholine (l-α-Phosphatidylcholine, also named soybean hosphatidylcholine, SPC) was supplied by Sigma Aldrich (St. Louis, MO, USA). Oleic Acid was acquired by Fluka Chemika (Buchs, Switzerland). Coll from Clostridium histolyticum type IA, Dulbecco’s Modified Eagle’s Medium—high glucose (DMEM), quercetin, thiazolyl blue tetrazolium bromide (MTT), Span 20^®, Nile Blue A and ascorbic acid were supplied by Sigma Aldrich (Steinheim, Germany). Fetal bovine serum (FBS) purchased from Biowest (Riverside, CA, USA). L-Glutamine was supplied by Lonza (Leuven, Belgium). Rutin was acquired from Extrasynthese (Genay, France). Methanol was purchased from António M. S. Cruz (Amadora, Portugal) and sodium chloride (NaCl) from José M. Vaz Pereira (Benavente, Portugal). Epigallocatechingallate (EGCG), N-[3-furyl-acryloyl]-Leu-Gly-Pro-Ala (FALGPA) were purchased from Panreac (Barcelona, Spain). Tricine buffer was acquired by VWR (Leuven, Belgium). Carbopol 940^® was purchased from Fagron (Barcelona, Spain). Folin reagent was supplied by Merck (Darmstadt, Germany). High performance liquid chromatography (HPLC) grade acetonitrile and formic acid were obtained from Chem-Lab NV (Zedelgem, Belgium). Milli-Q water (18.2 MΩ cm⁻¹ resistivity) was obtained from a Millipore-Direct Q3 UV system (Millipore^®, Burlington, MA, USA). All other chemicals were of analytical grade.

Methanol (MeOH, 99.8% LC–MS) and acetonitrile (CH₃CN, 99.9% LC–MS) were purchased from Fisher Scientific (Thermo Fisher Scientific, Waltham, MA, EUA). The ultra-pure water (18.2 MO.cm) was obtained from a Millipore-Direct Q3 UV system (Millipore, Billerica, MA, USA). Formic acid (HCOOH, 98% p.a.) was purchased from Merck (Darmstadt, Germany). Folin–Ciocalteu’s reagent, fluorescein sodium salt, AAPH (2,2-azobis(2-methylpropionamidine)dihydrochloride) and Trolox (6-hydroxy-2,5,7,8-tetramethyl chromane-2-carboxylic acid) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Standards of phenolic compounds, quercetin-3-glucoside (PubChem CID: 25203368) and gallic acid (PubChem CID: 370), were obtained from Sigma-Aldrich (St. Louis, MO, USA), and chlorogenic acid (3-O-caffeoylquinic acid–PubChem CID: 1794427) was purchased from Extrasynthese (Genay, Rhône-Alpes, France).

The LC system was a Waters® Alliance 2695 (Waters®) equipped with a quaternary pump, solvent degasser, auto sampler and column oven, coupled to a Photodiode Array Detector Waters 996 PDA (Waters®) scanning from 210 to 600 nm. The separation was performed on a reversed phase column LiChrospher® 100 RP-18 (10 x 4.0mm; Merck®, Germany) operating at 35 ^oC, using an injection volume of 10 μl. The mobile phase consisted of water [Ultra-pure water (18.2MΩ.cm) from a Millipore-Direct Q3 UV system (Millipore, USA)] with TFA 0.1% (v/v) (98% p.a.; Merck®, Germany) (A) and acetonitrile with TFA 0.1% (v/v) (B) at a flow rate of 0.30 ml/min. The following gradient was used: 70% of A as initial conditions, 70% A to 30% A for 7 min. A cleaning step was performed using 100% of acetonitrile for 1 min and finally the column was re-equilibrated with 70% of eluent A for 5 min.