Uplc q tof ms

The collection, normalization, and alignment of the MS dataset analyzed using the UPLC-Q-TOF MS were carried out using UNIFI version 1.8.2 (Waters Corp.). The peaks were collected using the peak-to-peak baseline noise as 1, a peak width at a 5% height of 1 s, noise elimination of 6, and an intensity threshold of 10,000. The collected data were aligned using a mass window of 0.05 Da and a retention time window of 0.2 min. Analyzer Pro application (Spectralworks Ltd., Runcorn, UK) was used to analyze the GC-MS data. The peaks were collected using an area threshold of 10,000, height threshold of 1, signal to noise ratio of 10, width threshold of 0.01, scan windows of 5, and smoothing of 5. The collected data were aligned with a retention time window of 0.1 min. All LC-MS and GC-MS data were normalized using the average mass intensity of the internal standards. The metabolites were identified based on the online databases (NIST 11 and Wiley 9 mass spectral libraries for GC-MS; ChemSpider database in UNIFI, METLIN database (www.metlin.scripps.edu), and human metabolome databases (www.hmdb.ca) for LC-MS), authentic standards, and retention indices (RIs) calculated using n-alkanes for GC-MS.

Identification and quantification of predominant polyphenolic metabolites were performed using the Quadrupole time-of-flight (QTOF) mass spectrometer (MS) UPLC-QTOF/MS (Waters, Milford, MA, USA, as described earlier by Managa et al. [3 (link)] and Ndou et al. [61 (link)] using 5 g sample of snap frozen Chinese cabbage and Nightshade leaves from oven drying, solar drying, microwave drying and freeze drying. The conditions for separation of the phenolic compounds are Ndou et al. [58 (link)]. The identification and quantification of the phenolic components were carried out using cocktail standards chlorogenic acid, catechin, luteolin, epicatechin and rutin, as previously described [3 (link),58 (link)], due to the to the unavailability of the calibration standards for all the compounds. The obtained data was processed using the established TargetLynx method to produce integrated peak areas for each compound as described in detail previously [3 (link),59 ].

Grape skin samples were lyophilized and ground under liquid nitrogen using a retsch‐mill (Retsch, Haan, Germany) with pre-chilled holders and grinding beads. For metabolite extraction, 40 mg of frozen skin powder was weighed and extracted in a 1-ml pre-cooled methanol:chloroform:water extraction solution (2.5:1:1 v/v) with ampicillin (1 mg ml⁻¹ in water) and corticosterone (1 mg ml⁻¹ in methanol) as internal standards as described by Hochberg et al. (2013) (link) and Degu et al. (2014) (link). Skin extracts were filtered (0.22 μm Millipore, MA, USA) and transferred to glass vials for analysis using ultra-performance liquid chromatography coupled to a quadrupole time‐of‐flight mass spectrometer (UPLC QTOF‐MS; Waters, MA, USA).
Chromatographic separation and the MS conditions were exactly as described previously (Hochberg et al., 2013 (link)).

Total GSH (reduced) and GSSG (oxidized) content was determined with UPLC-Q-TOF-MS (Waters Corp., Manchester, UK)72 (link). The relative content of amino acids implicated in water deficit tolerance; glycine (Gly), glutamine (Gln), glutamic acid (Glu), proline (Pro), leucine (Leu), serine (Ser), tryptophan (Trp) and valine (Val) was determined with a Thermo Scientific DSQ II GC/MS using a Factor Four Capillary VF-5ms column69 (link). Electrolyte conductance (EC) was determined as described previously10 (link)11 (link).

Grape skin samples were analyzed using Ultra Performance Liquid Chromatography coupled to a Quadrupole Time-of-Flight Mass-Spectrometer (UPLC QTOF-MS, Waters, MA, USA), following an extraction protocol for metabolite profiling as described in Weckwerth et al. (2004 (link)). Skin tissues were lyophilized and ground under liquid nitrogen using a RETSCH-mill (Retsch, Haan, Germany) with pre-chilled holders and grinding beads. The powder was weighed (40mg), and metabolites were extracted in a 1-ml pre-chilled methanol:chloroform:water extraction solution (2.5:1:1 v/v). Internal standards, i.e., 0.2 mg/ml ribitol in water, 1 mg/ml ampicillin in water, 1 mg/ml corticosterone in methanol, were subsequently added. The mixture was then briefly vortexed, and 100 μl of methanol was added; the mixture was then placed on a horizontal shaker for 10 min at 1000 rpm. The samples were later sonicated for 10 min (Elmasonic S30, Elma, Singen, Germany) and centrifuged for 10 min (20,817 × g, microcentrifuge 5417R, Eppendorf, Hamburg, Germany). The supernatant was decanted into new tubes, mixed with 300 μl of chloroform and 300 μl of MiliQ water (Millipore, MA, USA), vortexed for 10 s and then centrifuged at 20,817 × g for 5 min. Next, the water/methanol phase was separated, filtered (0.22 μm Millipore, MA, USA) and transferred to UPLC vials for analysis.

In this experiment, UPLC/Q-TOF-MS (Waters Co., United States) was used to characterize metabolic substances. UPLC separation was performed on the ACQUITY UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm, Waters Co., United States), with a column temperature of 45°C. The mobile phase consisted of water (A) and acetonitrile (B) (both containing 0.1% formic acid). The gradient elution program was as follows: 0–0.5 min, 1% B; 0.5–2 min, 1%–50% B; 2–9 min, 50%–99% B; 9–10 min, 99% B; 10–10.5 min, 99%–1% B; and 10.5–12 min, 1% B. The flow rate was 0.3 ml/min. The injection volume was 5 μL.
After separation, an electrospray ion source was used to detect and analyze mass spectrometry in positive and negative ionization modes. The ion source parameters were set as follows: a capillary voltage of 3.0 kV, drying gas temperature of 325°C, atomizing gas pressure of 310 kPa, drying gas flow of 0.26 ml/min, desolvation gas flow of 600 L/h, source temperature of 120°C, desolvation temperature of 350°C, and cone gas flow of 50 L/h.

Phytochemicals in the OHE were analyzed using UPLC-Q-TOF MS (Waters, Milford, MA, USA). The OHE was injected into an Acquity UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 µm; Waters) at a column temperature of 40 °C. The mobile phase consisted of water with 0.1% formic acid (FA) (A) and acetonitrile with 0.1% FA (B) at a flow rate of 0.35 mL/min for 10 min. The eluents were analyzed using a Q-TOF mass spectrometer with negative mode ESI. The scan range of TOF MS data was from 50 to 1500 m/z with a scan time of 0.2 s. The capillary voltage was set at 2.5 kV for the negative mode, while the sample cone voltage was 40 V. The desolvation flow rate was 900 L/h at a temperature of 400 °C, and the source temperature was set to 100 °C. Leucine-enkephalin ([M + H] = m/z 556.2771; [M − H] = m/z 554.2615) was used as a reference for lock mass at a frequency of 10 s. The MS/MS spectra were obtained using collision energy ramps from 20 to 45 eV. Major phytochemicals were identified by using the UNIFI scientific information system (Waters) with various LC/MS online databases.