The fermentation material was extracted repeatedly with EtOAc (4 × 4.0 L), and the organic solvent was evaporated to dryness under vacuum to afford 2.7 g of crude extract. The crude extract was fractionated by silica gel vacuum liquid chromatography (VLC) using petroleum ether–EtOAc–MeOH gradient elution. The fraction (107.5 mg) eluted with 6.5 : 1 petroleum ether–EtOAc was separated by Sephadex LH-20 column chromatography (CC) eluting with 1 : 1 CH2Cl2–MeOH and the resulting subfractions were combined and purified by semipreparative RP HPLC (Agilent Zorbax SB-C18 column; 5 μm; 9.4 × 250 mm; 47% CH3CN in H2O for 30 min; 2 mL min−1) to afford 2 (1.0 mg, tR 20.0 min). The fraction (165.8 mg) eluted with 3.5 : 1 petroleum ether–EtOAc was separated by reversed-phase silica gel column chromatography (CC) eluting with a MeOH–H2O gradient. The subfraction (18 mg) eluted with 40% MeOH–H2O was purified by semipreparative RP HPLC (Agilent Zorbax SB-C18 column; 5 μm; 9.4 × 250 mm; 37% CH3CN in H2O for 30 min; 2 mL min−1) to afford 3 (4.0 mg, tR 22.5 min). The subfraction (23 mg) eluted with 50% MeOH–H2O was purified by semipreparative RP HPLC (Agilent Zorbax SB-C18 column; 5 μm; 9.4 × 250 mm; 55% MeOH in H2O for 90 min; 2 mL min−1) to afford 4 (2.7 mg, tR 67.5 min) and 1 (3.4 mg, tR 82.5 min).
Agilent zorbax sb c18 column
Manufactured by Agilent Technologies
Sourced in United States, China, Japan
The Agilent Zorbax SB-C18 column is a reversed-phase liquid chromatography column designed for the separation and analysis of a wide range of analytes. It features a non-endcapped, high-purity silica-based stationary phase with a C18 alkyl functionality, which provides excellent retention and selectivity for a variety of polar and non-polar compounds.
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Lab products found in correlation
Agilent 1260 hplc system, by Agilent Technologies (2 mentions)
Cto 20a, by Shimadzu (2 mentions)
Agilent 1260 series hplc system, by Agilent Technologies (2 mentions)
Agilent 1100 hplc, by Agilent Technologies (2 mentions)
Ultimate 3000 system, by Thermo Fisher Scientific (2 mentions)
Agilent 1260 series, by Agilent Technologies (2 mentions)
Fluorescent detector, by Agilent Technologies (1 mentions)
Agilent 1200 hplc, by Agilent Technologies (1 mentions)
Triple tof 5600 1, by AB Sciex (1 mentions)
Lc 30ad hplc system, by Shimadzu (1 mentions)
Agilent 6530 q tof ms, by Agilent Technologies (1 mentions)
Sil 30ac, by Shimadzu (1 mentions)
Lc 30ad, by Shimadzu (1 mentions)
Dgu 20a, by Shimadzu (1 mentions)
Cbm 20a, by Shimadzu (1 mentions)
Alliance 2695 lc system, by Waters Corporation (1 mentions)
Agilent zorbax sb c18, by Agilent Technologies (1 mentions)
Diode array detector, by Agilent Technologies (1 mentions)
Q exactive, by Thermo Fisher Scientific (1 mentions)
Agilent 1260 infinity hplc system, by Agilent Technologies (1 mentions)
34 protocols using agilent zorbax sb c18 column
1
Isolation and Purification of Bioactive Compounds
2
Quantifying Bioactive Compounds in Coptis chinensis
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Dry powder samples (0.2 g) were extracted with 50 ml hydrochloric acid ethanol mixed liquor (1:100, v/v) for 30 min and sonicated for 30 min. Determination of the major bioactive components of C. chinensis was performed on an Agilent 1260 HPLC system (Agilent Technologies, Santa Clara, Calcium, USA) with an Agilent ZORBAX SB-C 18 column (250 mm × 4.6 mm, 5 mm, Agilent Technologies) at a column temperature of 30 °C and a flow rate of 1 mL/min. The mobile phase was acetonitrile—0.05 mol/L potassium dihydrogen phosphate solution (50:50, v/v), containing 0.1% sodium dodecyl sulfate, separated by equal-diameter elution. The detection wavelength was 345 nm. The contents of berberine, palmatine, cannine, coptidine, magnoline, and columbine were calculated by standard curves28 (link),51 (link). One-way analysis of variance was used to compare various BIA contents among roots in different years. If the variances were significantly different, Tamhane’s T2 test was used to perform post hoc analyses; otherwise, a least significant difference test was used. The results were considered significant at p < 0.05.
3
Extraction and Characterization of Raspberry Polyphenols
4
HPLC-QTOF/MS Protocol for Compound Analysis
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A Shimadzu LC30-AD HPLC system (Shimadzu, Kyoto, Japan) combined with a quadrupole time-of-flight mass spectrometer (Triple TOF 5600-1, AB SCIEX, Redwood City, CA, USA) was used. An Agilent Zorbax SB-C18 column (4.6 mm × 50 mm, 1.8 μm, Agilent Technologies, USA) with a column temperature maintained at 30 °C was employed. The mobile phase consisted of 0.1% formic acid (A) and acetonitrile (B), using a gradient elution of 10–20% B at 0–2 min, 20–22% B at 2–9 min, 22–40% B at 9–16 min, 40–70% B at 16–17 min, 70–100% B at 17–18 min, and 100% B at 18–19 min with an equilibrium for 4 min. The flow rate was set at 0.4 mL min−1 with an injection volume of 2 μL.
MS detection was performed using QTOF/MS in negative ionization mode with a DuoSpray ion source. The QTOF/MS was calibrated in high sensitivity mode and the automated calibration device system (CDS) was set to perform an external calibration every four samples using a calibration solution. The source parameters were optimized: collision voltage (CE), 50 eV; ion spray voltage floating (ISVF), 4500 V; temperature, 500 °C; nebulizing gas (GS1), 60 psi; heater gas (GS2), 60 psi; curtain gas, 35 psi. The MS was operated in full-scan TOF/MS (100–2000 amu) and MS/MS mode (100–2000 amu) through data-independent acquisition (DIA) in a single-run analysis.16 (link)
MS detection was performed using QTOF/MS in negative ionization mode with a DuoSpray ion source. The QTOF/MS was calibrated in high sensitivity mode and the automated calibration device system (CDS) was set to perform an external calibration every four samples using a calibration solution. The source parameters were optimized: collision voltage (CE), 50 eV; ion spray voltage floating (ISVF), 4500 V; temperature, 500 °C; nebulizing gas (GS1), 60 psi; heater gas (GS2), 60 psi; curtain gas, 35 psi. The MS was operated in full-scan TOF/MS (100–2000 amu) and MS/MS mode (100–2000 amu) through data-independent acquisition (DIA) in a single-run analysis.16 (link)
5
Accurate Mass Spectrometry Analysis
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Identification of mass spectrum was employed on an accurate mass spectrometer of Agilent 6530 Q-TOF-MS (Agilent Technologies, Palo Alto, CA, USA). Chromatographic separation was employed on an Agilent-ZORBAX SB-C18 column (250 mm × 4.6 mm, 5 μm, Agilent Technologies, Palo Alto, CA, USA), and the effluent of the HPLC mobile phase was split and guided into the electrospray ionization (ESI) source. Parameter conditions were performed as following: capillary voltage, 3500 V; nebulizer pressure, 50 psi; nozzle voltage, 1000 V; flow rate of drying gas, 6 L/min; temperature of sheath gas, 350 °C; flow rate of sheath gas, 11 L/min; skimmer voltage, 65 V; OCT1 RF Vpp, 750 V; fragmentor voltage, 135 V. The spectra data were recorded in the range of m/z 100–1000 Da in a centroid pattern of full-scan MS analysis mode. The MS/MS data of the selected compounds were obtained by regulating diverse collision energy (18–45 eV).
6
HPLC Separation of Analytes
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The HPLC chromatographic separation was achieved through a Shimadzu apparatus (Kyoto, Japan) with system controller (CBM-20A), pump (LC-30AD), autoinjector (SIL-30AC), online degasser (DGU-20A), and column heater (CTO-20A). An Agilent Zorbax SB-C18 column with the diameter of 2.1 × 100 mm (3.5 μM, Agilent, USA) was applied to separate the analytes. The temperature of column was 40°C. Analytes were equivalently eluted with 30:70 (v/v) acetonitrile-water containing 5 mM ammonium acetate (pH 4) and the flow rate was 0.3 ml/min. The analysis time of all analytes was 1.8 min, and the injection volume was 10 μl.
7
Quantitative Analysis of Limonin and Nimodipine
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Analyses were performed by an Alliance 2695 LC system (Waters, Milford, MA, USA) coupled with a triple-quadrupole tandem Quattro Micro mass spectrometer (Waters, Milford, MA, USA). Instrumental control is the Mass Lynx 4.1 software using for acquisition and processing of the data. The LC separation was performed on an Agilent Zorbax SB-C18 column (150 2.1 mm i.d. 5 mm, Agilent Technologies, Wilmington, DE, USA) by the mobile phase consisting of acetonitrile and deionized water (40:60, v/v) containing 10 mM at a flow rate of 0.8 mL/min with a security guard column (12.5 2.1 mm i.d. 5 mm, Agilent Zorbax SB-C18, DE, USA). The autosampler temperature was maintained at 15 °C. The total LC run time was 10 min with the column temperature kept at 30 °C.
The typical operating source condition for MS detector with an electrospray ionization (ESI) interface in negative ion mode. The detertion parameters were optimized as follows: capillary voltage, 2.7 kV; cone voltage, 45 V; source temperature, 110 °C; desolvation temperature, 350 °C; desolvation gas flow (nitrogen), 450 L/h; collision energy 28 V for limonin and 25 V for nimodipine; argon was used as the collision gas with the gas pressure of 3.0 × 10−3 mbar.
The typical operating source condition for MS detector with an electrospray ionization (ESI) interface in negative ion mode. The detertion parameters were optimized as follows: capillary voltage, 2.7 kV; cone voltage, 45 V; source temperature, 110 °C; desolvation temperature, 350 °C; desolvation gas flow (nitrogen), 450 L/h; collision energy 28 V for limonin and 25 V for nimodipine; argon was used as the collision gas with the gas pressure of 3.0 × 10−3 mbar.
8
Simultaneous Determination of Echinacoside and Acteoside
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To cross-validate the findings for the three processing techniques, an Agilent 1260 series HPLC system consisting of a degasser, a quaternary pump, an autosampler, a column oven, and a diode array detector (Agilent Technologies, Santa Clara, CA, USA) were employed for the simultaneous determination of echinacoside and acteoside in all a-, b- and c-type samples. An Agilent Zorbax SB-C18 column (250 mm×4.6 mm, particle size 5 μm, Agilent Technologies) protected by the corresponding guard column (10 mm×4.6 mm, particle size 5 μm) was selected to conduct chromatographic separations. The sample preparation protocol, mobile phase, and elution program followed the descriptions archived in Chinese Pharmacopeia (2010 Edition)3 with minor modifications. In brief, ultrasonic-assisted extraction was performed for 30 min using 50% aqueous methanol; 30% aqueous methanol was adopted as the mobile phase for isocratic elution at a flow rate of 1.0 mL/min, and the column oven was maintained at 30 °C. The detection wavelength was set at 330 nm, and the injection volume was set as 10 μL.
9
HPLC Analysis of Antioxidant Components
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The antioxidant components of the GMBC extract obtained under the optimal condition were investigated by high-performance liquid chromatography (HPLC) according to our previous method [12 (link)]. In brief, analysis of HPLC was conducted with a Prominence Modular HPLC system, which is made up of a binary pump, an online degasser, an auto-sampler, and a photodiode array detector. An Agilent Zorbax SBC18 column (4.6 mm × 150 mm, 3.5 µm) (Agilent Technologies, Santa Clara, CA, USA) was employed to separate antioxidant ingredients from the extracts. The mobile phase was made up of solution A (2.5% formic acid aqueous solution) and solution B (100% methanol). The gradient elution (the flow rate, 0.8 mL/min; the injection volume, 20 µL) was performed as follows: 0–15 min, 5%–30% B; 15–40 min, 30%–40% B; 40–45 min, 40%–50% B; 45–50 min, 50%–95% B; 50–60 min, 95% B; 60–65 min, 95%–5% B; 65–75 min, 5% B. Catechin and gallic acid were detected at 280 nm, p-coumaric acid, vitexin, and isovitexin were detected at 320 nm, and rutin was detected at 350 nm. The retention time and spectra of antioxidants were compared with the standard compounds, and were quantified according to the peak areas. The result was showed as g/kg DW (dry weight) of GMBC.
10
Enzymatic Activity of RchUGT169 Evaluated
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Using UDP-Glc as a glycosyl donor and quercetin or kaempferol as the receptors, the reaction system (100 μL) to verify RchUGT169 protein activity was as follows: experimental groups (4 μL UDP-Glc, kaempferol or quercetin 1 μL, RchUGT169 protein was dissolved with 100 μL 1 × Hepes buffer) and the control group (4 μL UDP-Glc, kaempferol or quercetin 1 μL, RchUGT169 protein (inactivated at 90 °C) was dissolved with 100 μL 1 × Hepes buffer). The above solutions were mixed and reacted at 25 °C, 1300 rpm for 4 h in a metal bath, respectively, and the supernatants were filtered with 0.22 um filter membrane for LC-MS/MS (Thermo Scientific Q Exactive, Waltham, MA, USA) detection at 350 nm. The chromatographic column was Agilent ZORBAX SB-C18 column (250 × 4.6 mm, 5 μm) (Agilent, Santa Clara, CA, USA), the column temperature was 30 °C, the injection volume was 2 μL, the mobile phase A was 0.1% formic acid aqueous solution (v/v), and the mobile phase B was 100% acetonitrile. The gradient elution procedure was as follows: 0–2 min, 30% B; 2–20 min, 30% B; 20–25 min, 100% B; 25–30 min, 30% B. The experimental conditions of mass spectrometry were as follows: the ion source was ESI, the negative ionization mode was used, and the acquisition mode was full scan, auto MS/MS.
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