As 2057 plus intelligent sampler

Phenolic acid analysis was performed following a previous method after minor modifications [19 (link)]. A Jasco HPLC instrument (Tokyo, Japan), equipped with a quaternary gradient pump Jasco PU-2089, an autosampler Jasco AS-2057 Plus Intelligent Sampler and two detectors, a Jasco UV/Vis MD-910 PDA detector and a Jasco FP-2020 Plus Fluorescence detector, was used. The column was a C18 Poroshell 120 (Agilent technologies, Santa Clara, CA, USA), 2.7 μm, (4.6 mm × 150 mm), operating at 35 °C with a flow of 0.8 mL/min. Elution solvents were 2% acetic acid in HPLC grade water (Eluent A) and 2% acetic acid in HPLC grade acetonitrile (Eluent B). Gradient elution was as follow: from 98% to 95% A in 10 min, 95% to 90% A in 7 min, 90 to 82% A in 6 min, 82% to 80% A in 3 min, 80% to 70% A in 3 min, 70% to 50% A in 3 min, 50% to 0% A in 4 min and 98% A in 1 min. Quantification of phenolic compounds was carried out using an external calibration curve obtained by injecting solutions of standard compounds at known concentrations and plotting peak areas vs. concentrations. The amount of tartrate esters of caffeic, coumaric and ferulic acids and Grape reaction Product GRP were expressed as the respective hydroxycinnamic acid.

HPLC analyses were performed on a Jasco system (Gross-Umstadt, Germany) equipped with a PU-2080 Plus Intelligent HPLC Pump, a DG-2080-53 3-Line Degasser, a LG-2080-02 Ternary Gradient Unit, a AS-2057 Plus Intelligent Sampler, a Column-Thermostat Jetstream Plus, and a MD-2010 Plus Multiwavelength Detector. Proanthocyanidins were separated on a reversed phase Aqua 5 µ C-18, 125 Å, 250 × 4.6 mm column (Phenomenex, Aschaffenburg, Germany) at 280 nm protected by a guard column of the same material. The same eluent and gradient were used as in the polyphenol LC-MS/MS analysis, with a flow rate of 0.5 mL/min at 25 °C. Seed samples were diluted in acetonitrile/water (1:9, v/v; approximately 5.5 mg/mL). Briefly, 20 µl from all samples was injected three times, and (-)-epicatechin standard solutions in the range of 0.5 to 1000 mg/L were injected twice.

HPLC–PDA quantification of polymeric compounds were performed on a Primesep B2, 5 µ, 150 mm × 2.1 mm column (SIELC, Wheeling, WV, USA) using a PU-2080 Plus Intelligent HPLC pump, a DG-2080-50 3-line Degasser, a LG-2080-02 Ternary Gradient Unit, an AS-2057 Plus Intelligent Sampler, a CO-2067 Plus Intelligent Column Oven, a MD-2010 Plus Multiwavelength Detector, and a LC-NetII/ADC Transmitter (Jasco, Gross-Umstadt, Germany). The quantification was carried out with solvent systems A (5% aqueous formic acid) and B (acetonitrile) at a flow rate of 0.2 and 0.4 mL/min and 40 °C using the following gradient: 0 min, 0% B; 20 min, 0% B; 60 min, 15% B; 65 min, 50% B; 70 min, 50% B; 75 min, 100% B; 80 min, 100% B; 85 min, 0% B; 100 min, 0% B. The quantification was carried out with catechin as standard (0.01–1 mg/mL, LOQ 0.09 mg/mL, LOD 0.002 mg/mL) at λ = 280 nm.