For the extraction and determination of carotenoids and chlorophylls, a protocol described earlier was followed [48 (link)]. The compounds were separated with an ACQUITY UPLC BEH RP C18 column (1.7 µm, 2.1 mm × 100 mm; Waters Corp.) at 32 °C. The elution solvents were ACN:MeOH (7:3, v/v) (A) and 0.1% formic acid (B). All incubations were done in triplicate. The results are expressed as milligrams per kilogram DW.
Acquity uplc beh c18 rp column
Manufactured by Waters Corporation
Sourced in United States
The Acquity UPLC BEH C18 RP column is a reversed-phase high-performance liquid chromatography (HPLC) column designed for ultra-high-performance liquid chromatography (UPLC) applications. It features a bonded C18 stationary phase and a bridged-ethyl hybrid (BEH) particle technology, providing high-efficiency separation and resolution of a wide range of analytes.
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Lab products found in correlation
Acquity uplc system, by Waters Corporation (3 mentions)
Acquity synapt g2 q tof tandem mass spectrometer, by Waters Corporation (2 mentions)
Millex simplicity filter, by Merck Group (1 mentions)
Thermo uhplc u3000 pump system, by Thermo Fisher Scientific (1 mentions)
Hplc grade acetonitrile, by Thermo Fisher Scientific (1 mentions)
Formic acid, by Thermo Fisher Scientific (1 mentions)
Methanol, by Thermo Fisher Scientific (1 mentions)
Masslynx version 4, by Waters Corporation (1 mentions)
Masslynx v4, by Waters Corporation (1 mentions)
6 protocols using acquity uplc beh c18 rp column
1
Carotenoid and Chlorophyll Extraction
2
Extraction and Analysis of Garlic Carotenoids
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Sample extraction was performed as described by Lachowicz et al. [13] . Freeze-dried different parts of wild garlic (~ 0.35 g) were mixed with 5 mL of hexane:acetone:methanol (2:1:1), shaken at 20 °C for 30 min and 40 kHz. Next, the slurry was centrifuged at 19,000g for 10 min, and the supernatant was evaporated to dryness. The pellet was re-extracted using 2 mL of 100% methanol, filtered through a hydrophilic PTFE 0.20 µm membrane (Millex Simplicity Filter, Merck, Darmstadt, Germany), and used for analysis.
For the extraction of carotenoids, a protocol similar to that described earlier was followed [14] . Compounds were separated with an ACQUITY UPLC BEH RP C18 column (1.7 µm, 2.1 mm × 100 mm, Waters Corp., Milford, MA, USA) at 32 °C. The elution solvents were ACN:MeOH (7:3, v/v) (A) and 0.1% formic acid (B). Samples (10 µL) were eluted according to the linear gradient described by Delphino-Rius et al. [15] . The runs were monitored at 450 and 650 nm. The PDA spectra were measured over the wavelength range of 200-700 nm in steps of 2 nm. The retention times and spectra were compared to those of the authentic standards. All incubations were done in triplicate. The results were expressed as milligram per kilogram of dry matter (mg/kg dm).
For the extraction of carotenoids, a protocol similar to that described earlier was followed [14] . Compounds were separated with an ACQUITY UPLC BEH RP C18 column (1.7 µm, 2.1 mm × 100 mm, Waters Corp., Milford, MA, USA) at 32 °C. The elution solvents were ACN:MeOH (7:3, v/v) (A) and 0.1% formic acid (B). Samples (10 µL) were eluted according to the linear gradient described by Delphino-Rius et al. [15] . The runs were monitored at 450 and 650 nm. The PDA spectra were measured over the wavelength range of 200-700 nm in steps of 2 nm. The retention times and spectra were compared to those of the authentic standards. All incubations were done in triplicate. The results were expressed as milligram per kilogram of dry matter (mg/kg dm).
3
Quantitative Proteomics of Arabidopsis
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The labelled samples were fractionated using a Thermo UHPLC U3000 Pump system (Thermo Fisher Scientific, San Jose, CA) with an ACQUITY UPLC BEH C18 RP column (1.7 μm particle size, 2.1 × 100 mm; Waters, USA). Detailed specific parameters for the LC-MS/MS analysis are given in our previous research.31,32 (link) The raw MS/MS files were processed using the Proteome Discoverer Software 1.4 (ESI Table S2 and ESI Fig. S2† ). Protein identification was performed using the Sequest HT engine against the uniprot Arabidopsis thaliana database. The search parameters were as follows: trypsin was selected as the enzyme, with the tolerance set at one missed cleavage, a peptide allowance of 10 ppm, and an MS and MS/MS allowance of 0.02 Da. To be identified as important differentially abundant proteins (DAPs), a protein needed to contain at least one unique peptide with a p-value less than 0.05 and a fold change greater than 1.5 or less than 0.67.32 (link) Identified proteins were annotated with their biological functions according to KEGG (http://www.genome.jp/kegg/ ) and the literature. Information on the DAPs was obtained from the Universal Protein Resource (http://www.uniprot.org/ ). Pathway enrichment analysis was performed using KOBAS 3.0 (http://kobas.cbi.pku.edu.cn/ ).
4
UPLC-TOF-MS Analysis of Bioactive Compounds in WFC
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WFC tablet was kindly provided by Huqingyu-tang Pharmaceutical Co., Ltd. (Hangzhou, China). Quality and quantity analyses of the aqueous extract were performed with UPLC TOF-MS. HPLC-grade acetonitrile, methanol, and formic acid were purchased from Fisher Scientific (Santa Clara, USA). Naringin, ginsenoside Rb1, and oridonin were identified in WFC by UPLC TOF-MS. The following conditions were used to analyze naringin, ordionin, and ginsenoside Rb1: system, Acquity UPLC system (Waters, USA), which consists of a solvent degasser, a binary pump, an auto-sampler and a column oven; column, Acquity UPLC BEH C18 RP column (1.7 μm, 100 mm × 2.1 mm i.d.; Waters, USA); mobile phase A, 0.1% formic acid in water; mobile phase B, 100% acetonitrile; flow rate, 0.3 mL/min; wavelengths, 210 nm for ginsenoside Rb1, 254 nm for ordionin and 280 nm for naringin; injection volume, 10 μL; MS/MS detector, Acquity Synapt G2 Q-TOF tandem mass spectrometer connected to the UPLC system by an ESI interface and controlled by MassLynx version 4.1 (Waters, UK). Samples were analyzed in the positive model. Data were collected and analyzed by Waters MassLynx version 4.1.
5
UPLC-QTOF-MS Analysis of PASE Compounds
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The analysis of PASE was performed on an Acquity UPLC system (Waters, United States) combined with an Acquity Synapt G2 QTOF tandem mass spectrometer (Waters, United Kingdom). An Acquity UPLC BEH C18 RP column (1.7 μm, 100 mm × 2.1 mm i.d.; Waters, United States) was employed for the chromatographic separation with the column temperature at 45°C. The mobile phase consisted of 0.1% formic acid in deionized water (mobile phase A) and acetonitrile (mobile phase B) at a flow rate of 0.3 ml/min with the following gradient: 0–4 min, 15% B; 4–7 min, 15–17% B; 7–9 min, 17–23% B; 9–14 min, 23–50% B; 14–28 min, 50–95% B, and 28–30 min, 5% B for equilibration of the column. In the MS analysis, the ESI source was operated in both positive (ESI+) and negative (ESI-) ionization mode with the following parameters: capillary voltage, −2.5 kV (ESI−) or +3 kV (ESI+); sample cone, 30 V; extraction cone, 4.0 V; source temperature, 120°C; desolvation temperature, 350°C; cone gas (nitrogen) flow, 50 L/h; and desolvation gas (nitrogen) flow, 600 L/h. Instrumental control and data collection and processing were conducted by MassLynx V4.1 software (Waters Corp., Milford, MA, United States).
6
UPLC Chromatographic Separation Protocol
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Chromatographic separations were performed on an Acquity UPLC system (Waters, USA) equipped with a binary solvent delivery system and an autosampler. The extracts were separated using an Acquity UPLC BEH C 18 RP column (1.7 µm, 100 mm × 2.1 mm i.d.; Waters, USA) in which the column temperature was maintained at 45°C to avoid excessive column pressure. The mobile phase consisted of 0.1% formic acid in deionized water (mobile phase A) and acetonitrile (mobile phase B). Separation was conducted with the following gradient elution at a flow rate of 0.3 mL/min: 0-1 min, 5% B; 1-4 min, 5-15% B; 4-5 min, 15-17% B; 5-7 min, 17% B; 7-9 min, 17-23% B; 9-14 min, 23-50% B; 14-23 min, 50-65% B; 23-28 min, 65-95% B, and 28-30 min, 5% B for equilibration of the column. The injection procedure was carried out for 1 min. An aliquot of 5 µL of sample solution was injected for analysis.
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