Agilent 1200 system

Targeted measurement of phenolic compounds in stored wines were conducted with a minimal extraction procedure. After overnight thawing in 4 °C dark room, wine samples were centrifuged (9335 g, IEC Micromax Centrifuge) for 5 min, filtered (0.45 µm cellulose syringe filter), and 5 µL of sample was directly injected into the reversed-phase Kinetex C₁₈ column (100 × 4.6 mm i.d., 2.6 µm pore size, 20 °C; Phenomenex, Torrance, CA, USA) on an Agilent 1200 system (Agilent Technologies, Palo Alto, CA, USA) connected to a diode-array detector. Solvents include 10% (v/v) formic acid (Merck, Darmstadt, Germany) in acetonitrile (solvent A; VWR International, Monroeville, PA, USA); and 10% (v/v) formic acid in deionized water (solvent B). Gradient elution using both solvents ran at a flowrate of 1 mL/min in the following conditions: 0–12.5 min, 96.7% solvent B; 12.5–13.5 min, 64.3% solvent B; 13.5–14.5 min, 96.7% solvent B; and 14.5–20 min, 96.7% solvent B. Phenolic compounds (i.e., anthocyanins, flavonols, flavanols, stilbenes, hydroxybenzoic, and hydroxycinnamic acids) were identified and quantified at 520, 360, 325, and 280 nm based on the retention times of the known standards (≥95% purity) and the external calibration curve of the respective authentic standard diluted in methanol. Results were then expressed as mg phenolic per liter of wine. Four sample replicates were analyzed.

The chromatographic measurements were performed using a complete HPLC Agilent 1200 system using a C18 Eclipse XDB column (4.6 × 150 mm, 5 μm). The HPLC system was coupled to a mass spectroscopy detector, Agilent 6110 single quadrupole (Agilent Technologies, CA, USA). The analytical mobile phase consisted of 0.1% acetic acid in water (solvent A) and 0.1% acetic acid in acetonitrile (solvent B). The compounds were separated using gradient elution with 0.5 mL/min flow at 25 °C for 30 min as follows: 5% solvent B for 2 min, increased to 40% until min 18 min, increased to 90% until 20 min, maintained at 90% for 4 min, decreased to 5% until 25 min, followed by another 5 min at 5 % for reconditioning. Chromatograms were recorded at 280 and 340 nm. Mass spectra were acquired in the positive ESI mode: 3000 V, 300 °C, 8 L/min nitrogen flow, m/z:100−1000, full-scan. Data acquisition was achieved using the ChemStation software (Agilent Technologies). The results were expressed as µg catechin equivalents (CE) /mL extract.

The high-performance liquid chromatography (HPLC) system comprised of Agilent 1200 system, autosampler, quaternary pump, solvent delivery mode, and column compartment (Agilent Technologies, Germany). Detector utilized an Agilent 6420 triple quad mass spectrometer (TQ-MS) which was coordinated by MassHunter software. The screw-capped (PTFE/silicon) total recovery 1-ml autosampler vial, 12 millimeter (mm) was used. The injection volume was 20 μL [40 (link)]. Chromatograms for simvastatin are shown in Fig 1.

Detection of α-galactosidase activity detection was evaluated using pNPG as substrate [21 (link)]. The reaction solution was terminated by adding Na₂CO₃ and filtered through a 0.22-μm membrane for high-performance liquid chromatography (HPLC) analysis. An Agilent 1200 system (Agilent Technologies, Palo Alto, CA, USA) with NH₂ column (Thermo Scientific, Sunnyvale, CA USA) was employed to detect the carbohydrates content. The carbohydrate concentration was calculated according to peak areas and retention time. The amount of enzyme releasing 1 μM p-nitrophenol per min at 45 °C and in buffer of pH 4.5 was defined as one unit of α-galactosidase activity (U). Furthermore, the α-galactosidase activity based on other three different substrates, including stachyose, raffinose and melibiose, were assessed. Three independent assays were carried out and the average values were calculated.

The dissolved lipids were eluted from the SPE cartridge with 2 mL of 70:30 acetonitrile: isopropanol into pre-combusted collection vials that were treated with the antioxidant BHT to prevent autoxidation of the samples. The samples were immediately transferred from the collection vials into HPLC vials and capped under argon. The samples were stored at −80 °C until mass spectrometric analysis. Within a week of extraction, the dissolved lipidome samples were analyzed using reverse-phase HPLC (Agilent 1200 system; Agilent, Santa Clara, CA, USA) paired with high-resolution, accurate-mass (HRAM) data from a Thermo Exactive Plus Orbitrap mass spectrometer (ThermoFisher Scientific, Waltham, MA, USA). The chromatographic method used a Xbridge C8 column (Waters, Milford, MA, USA) as the stationary phase, 18 MΩ water as eluent A, 70:30 acetonitrile: isopropanol as eluent B, and ammonium acetate and acetic acid as the adduct-forming additives [74 (link)]. The gradient began with a 1-min isocratic hold of 45% B and shifted to 99% B over 25 min with a flow rate of 0.4 mL min⁻¹. Full scan data (m/z 100–1500) were collected in negative mode at a mass resolution of 170,000.