Analysis was performed using an Agilent 1290 Infinity UPLC chromatography coupled to an iFunnel quadrupole time of flight (QTOF) Agilent 6550 spectrometer (Agilent Technologies, CA, USA). Samples were analyzed by a Synergi Hydro-RP C18 (150 × 1 mm, 4 µm) column (Phenomenex, Torrance, USA) that was heated to 50 °C in the column oven. The mobile phases consisted of acetonitrile with 0.1% (v/v) formic acid as solvent B and water with 0.1% (v/v) formic acid as solvent A. Sample analysis was initially performed by 1% of solvent B for 2 min. Then, a linear gradient of solvent B from 1 to 80% on 8 min was observed. Finally, 98% of solvent B was held for 2 min and then initial conditions for 3 min to allow reconditioning of the system. The flow rate used was 0.4 mL/min over 15 min chromatogram total run time. Electrospray positive mode was used for these experiments. To correct mass drifts during the acquisition, a reference standard of phthalic anhydride, purine and hexakis (1H, 1H, 3H-tetrafluoropropoxy) phosphazine was used. The samples are injected randomly and QC sample was injected every eight samples to correct instrumental drift.
Synergi hydro rp c18
Manufactured by Phenomenex
Sourced in United States
Synergi Hydro-RP C18 is a reversed-phase liquid chromatography column designed for the separation and analysis of a wide range of polar and non-polar compounds. It features a spherical silica-based stationary phase with a carbon-loaded C18 bonded phase. The column provides high-efficiency separations with excellent peak shape and resolution.
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3 protocols using synergi hydro rp c18
1
UPLC-QTOF Analysis of Metabolites
2
Metabolomic Sample Preparation for LC-MS
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Samples preparation for LC-MS metabolomics analysis followed the previous study (Li et al., 2015 (link)). Briefly, cells grown at the 24 or 48 h were centrifuged at 8,000 × g for 5 min at 25°C (Eppendorf 5430R, Hamburg, Germany). The supernatant was discard. The sediment was resolved in 900 µL of solution 1 (80:20 MeOH/H2O, stored at −80°C), quickly frozen in liquid nitrogen, and thawed on dry ice to release metabolites (Park et al., 2011 (link)). The cells were frozen-thrawed for three times to release the whole metabolites, and the supernatants were collected by centrifugation at 150,00 × g for 5 min at 4°C (Park et al., 2011 (link)). The left cell debris were then re-suspended in solution 1 and the above extraction process was repeated. The twice supernatants were mixed and stored at −80°C until LC-MS analysis. An Agilent 1,260 series binary HPLC system (Agilent Technologies, Waldbronn, Germany) using a SYnergi Hydro-RP (C18) 150 mm × 2.0 mm I.D., 4 μm 80 Å 549 particles column (Phenomenex, Torrance, CA, United States), coupled to an Agilent 6410 550 triple quadrupole mass analyser equipped with an electrospray ionization source was used for LC-MS analysis. Data processing and statistical analysis were conducted according to the method described previously (Wang et al., 2019 (link)).
3
Quantification of Wine Anthocyanins by HPLC-DAD-MS
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The anthocyanins in wine were determined by high performance liquid chromatography (LC-20AT, Shimadzu, Japan) and photodiode array detector (SPD-M20A, Shimadzu, China). The chromatographic column was determined by Synergi Hydro-RP C18 (250 × 4.6 mm, 4 µm, Phenomenex, Torrance, CA, USA). The anthocyanins with unknown structure were qualitatively identified by mass spectrometry before quantifying. Finally, the concentration of each anthocyanin was expressed as the equivalent of dimethyl-3-O-glucoside.
Based on the method of Yang et al. [21] (link), configuring the mobile phase A (formic acid:acetonitrile:water = 2.5:10:80, V/V/V) and mobile phase B (formic acid:acetonitrile:water = 2.5:50:40, V/V/V). The gradient elution procedure of mobile phase B was as follows: 0 ∼ 45 min: 0 %∼35 %; 45 ∼ 46 min: 35 %∼100 %; 46 ∼ 50 min: 100 %; 50 ∼ 51 min: 100 %∼0%; 51 ∼ 55 min: 0 %. The flow rate was set at 1 ml/min and the detection wavelength was set at 520 nm. The column temperature was 35 °C, the injection volume was 20 µL, the concentration range of the standard curve of anthocyanins was 0.27 ∼ 400 mg/L and the coefficient of determination was 0.9999. The anthocyanins were qualitatively analyzed according to their retention time, maximum absorption wavelength and experimental anthocyanin mass spectrometry database.
Based on the method of Yang et al. [21] (link), configuring the mobile phase A (formic acid:acetonitrile:water = 2.5:10:80, V/V/V) and mobile phase B (formic acid:acetonitrile:water = 2.5:50:40, V/V/V). The gradient elution procedure of mobile phase B was as follows: 0 ∼ 45 min: 0 %∼35 %; 45 ∼ 46 min: 35 %∼100 %; 46 ∼ 50 min: 100 %; 50 ∼ 51 min: 100 %∼0%; 51 ∼ 55 min: 0 %. The flow rate was set at 1 ml/min and the detection wavelength was set at 520 nm. The column temperature was 35 °C, the injection volume was 20 µL, the concentration range of the standard curve of anthocyanins was 0.27 ∼ 400 mg/L and the coefficient of determination was 0.9999. The anthocyanins were qualitatively analyzed according to their retention time, maximum absorption wavelength and experimental anthocyanin mass spectrometry database.
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