The flavonoid content of CSOL was analyzed using the Agilent 1260 LC system with a diode array detector-fluorescence detector and a C-18 column (150×4.6 mm; particle size, 5 µm) (Agilent Technologies, Inc.) with the injection volume of 10 µl and the flow rate of 1 ml/min by elution containing 98% methanol and 2% ultrapure water at 20°C. The standards were obtained from Shanghai Yuanye Bio-Technology Co. Ltd.
Agilent 1260 lc system
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The Agilent 1260 LC system is a high-performance liquid chromatography (HPLC) instrument designed for analytical applications. It consists of modular components that can be configured to meet specific laboratory needs. The system's core function is to separate, identify, and quantify various chemical compounds in a sample through the process of liquid chromatography.
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Lab products found in correlation
Kinetex c18 hplc column, by Phenomenex (3 mentions)
Eclipse xdb c18 column, by Agilent Technologies (2 mentions)
Luna c18 2, by Phenomenex (2 mentions)
Topspin 2, by Bruker (2 mentions)
Agilent 6530 accurate mass q tof ms, by Agilent Technologies (2 mentions)
Model 6540, by Agilent Technologies (2 mentions)
4000 qtrap mass spectrometer, by Thermo Fisher Scientific (2 mentions)
C18 column, by Agilent Technologies (1 mentions)
Agilent 6460 triple quadrupole ms, by Agilent Technologies (1 mentions)
Api 4000 qtrap mass spectrometer, by AB Sciex (1 mentions)
Luna c18 2 hst column, by Phenomenex (1 mentions)
Gc buffer, by Takara Bio (1 mentions)
Primestar hs kit, by Takara Bio (1 mentions)
Securityguard c18 column, by Phenomenex (1 mentions)
Calibrant solution a, by Agilent Technologies (1 mentions)
Agilent 6530 accurate mass qtof spectrometer, by Agilent Technologies (1 mentions)
C18 reversed phase hplc column, by Phenomenex (1 mentions)
Masshunter software, by Agilent Technologies (1 mentions)
Chemstation software, by Agilent Technologies (1 mentions)
Kinetex phenyl hexyl column, by Phenomenex (1 mentions)
35 protocols using agilent 1260 lc system
1
Quantifying Flavonoids in CSOL
2
HPLC Analysis of Flavonoids and Phenolic Acids
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The HPLC analysis was analyzed using an Agilent 1260LC system (Agilent Technologies, Santa Clara, CA, USA) [24 (link)]. Before injection, all samples were filtered by 0.22 µm nylon syringe filters. The C18 reversed-phase analytical column (250 mm × 4.6 mm, 5 µm, Greenherbs Science and Technology, Beijing, China) was maintained at 25 °C. Mobile phase A consisted of water containing 0.1% formic acid, and mobile phase B consisted of acetonitrile. The liquid chromatography gradient was as follows: 2–8% B in 0–12 min, 8–13% B in 12–15 min, 13–18% B in 15–30 min, 18–30% B in 30–50 min, 30–50% B in 50–60 min, 50–70% B in 60–70 min, 70–90% B in 70–80 min, 90–100% B in 80–85 min, and 100–2% B in 85–90 min. Each filtered 10 μL sample was determined in the UV scan range of 200–400 nm. The compounds were identified and quantified by comparing the retention times and peak areas of the authentic flavonoid and phenolic acid standards (gallic acid, catechin, chlorogenic acid, epicatechin, dihydromyricetin, and epicatechin gallate). For detailed information, refer to Supplementary Table S1 .
3
Photochemical Synthesis of N-acetyl-3-iodo-L-tyrosine
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Sample solutions (total volume 400 μL) contained DMCT or DOTC (100 µM), KI (400 mM) and N-acetyl-L-tyrosine ethyl ester (10 mM) in PB buffer (pH 7.4, containing 10% methanol) were irradiated by UVA light (365 nm) for DOTC or with blue light (415 nm) for DMCT with magnetic stirring. An aliquot of solution (50 μL) was removed at different time point (30 mins, 60 mins, 120 mins) and centrifuged at 4000 rpm. It was necessary to use relatively large fluences of light in order to get enough product to allow measurement of the peak area. The supernatants were collected for the LC-MS analysis. The LC-MS analyses were performed on an Agilent 1260 LC system equipped with a triple-quad mass spectrometer. The LC conditions were: column: C18, 2.1 × 50 mm, 1.8 μm; elution gradient: solution A = acetonitrile, solution B = 10 mM ammonium acetate in water, 2% −> 100% of A over 6 min with a flow rate of 0.2 mL/min; ionization mode: negative; injection volume: 5 uL. The mass of the molecular ions of N-acetyl-3-iodo-L-tyrosine ethyl ester was 37726 (link).
4
Sialylated N-Glycan Profiling by LC-MS/MS
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Dried sialylated N-glycans were reconstituted by 50 μl of water and then analyzed using Agilent 1260 LC system coupled to an Agilent 6460 triple quadrupole MS (Agilent Technologies, Santa Clara, CA). A Thermo Hypercarb™ column (3 mm i.d., 100 mm length, and 3 µm particle size) was used for LC separation. Upon injection of 2 μl of sample, sialylated N-glycans were chromatographically separated for 45 min using binary gradient consisting of solvent A of 3% ACN with 0.1% FA (v/v) and solvent B of 90% ACN with 0.1% FA (v/v) at a flow rate of 0.3 ml/min. The LC gradient used was as follows: 0–0.25 min, 7%, 0.3 ml/min B; 0.25–10 min, 7%–15%, 0.3 ml/min B; 10–25 min, 15%–40%, 0.3 ml/min B; 25–30 min, 40%–100%, 0.3 ml/min B; 30–35 min, 100%, 0.3 ml/min B; 35–35.1 min, 100%–7%, 0.3 ml/min B; and 35.1–45 min, 7%, 0.3 ml/min B. MS was operated in the positive mode. The first and third quadrupoles were operated at unit resolution. The optimal parameters used were as follows: dry gas temperature 250°C, dry gas flow 10 l/min, nebulizer pressure 30 psi, sheath gas temperature 300°C, sheath gas flow 12 l/min, and capillary voltage 4,000 V. MRM transitions monitored for the study are listed in Supplementary Table S2 .
5
Quantification of Lignans in Herbal Extracts
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SFES and SEES were analyzed by HPLC [23 ]. Using an Agilent 1260 LC system (Agilent, Santa Clara, California, USA) equipped with a G1314F VWD detector and an Eclipse XDB-C18 column (4.6 mm × 250 mm, 5 μm). The column temperature was 30°C, the detector wavelength was set at 220 nm, and the flow rate was 1.0 mL/min. Ultrapure water and acetonitrile were used as Mobile Phase A and B, respectively. The gradient elution was programmed as follows: 0–20 minutes, 50% A; 20–30 minutes, 50–40% A; 30–40 minutes, 45–25% A; 40–50 minutes, 25% A; 50–60 minutes, 25–35% A; 60–65 minutes, 35–50% A.
The mixed reference solution was prepared with acetonitrile at concentrations of 120 μg/mL (schisandrol A), 45 μg/mL (schisandrol B), 30 μg/mL (schisantherin A), 45 μg/mL (schisandrin A), 120 μg/mL (schisandrin B) and 30 μg/mL (schisandrin C). Standard curves of the six lignans were established by six different injection volumes (1 μL, 2 μL, 6 μL, 10 μL, 16 μL, and 20 μL) of the mixed reference solution. The solution was filtered through a 0.45-μm membrane before liquid chromatogram (LC) analysis.
The sample solutions of SFES and SEES were prepared by accurately weighing 0.3 g extract and transferring into a 10-mL volumetric flask with acetonitrile. The solution was filtered through a 0.45-μm membrane before LC. The injection volume of SFES was 5 μL, while the injection volume of SEES was 10 μL.
The mixed reference solution was prepared with acetonitrile at concentrations of 120 μg/mL (schisandrol A), 45 μg/mL (schisandrol B), 30 μg/mL (schisantherin A), 45 μg/mL (schisandrin A), 120 μg/mL (schisandrin B) and 30 μg/mL (schisandrin C). Standard curves of the six lignans were established by six different injection volumes (1 μL, 2 μL, 6 μL, 10 μL, 16 μL, and 20 μL) of the mixed reference solution. The solution was filtered through a 0.45-μm membrane before liquid chromatogram (LC) analysis.
The sample solutions of SFES and SEES were prepared by accurately weighing 0.3 g extract and transferring into a 10-mL volumetric flask with acetonitrile. The solution was filtered through a 0.45-μm membrane before LC. The injection volume of SFES was 5 μL, while the injection volume of SEES was 10 μL.
6
LC-MS/MS Analysis of Azaspiracids
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LC–MS/MS was performed on an Agilent 1260 LC system (Agilent Inc., Palo Alto, CA, USA) equipped with a binary pump, degasser, column oven (25 °C), and autosampler (6 °C). LC eluent was interfaced to an API4000 QTRAP mass spectrometer using a Turbospray ionization source (Sciex, Concord, ON, Canada), all controlled by Analyst 1.6.2 software. LC separations were on a Luna C18(2)HST column (50 × 2 mm, 2.7 µm; Phenomenex, Torrance, CA, USA) using gradient elution (0.3 mL/min) with a binary mobile phase of water (A) and 95% MeCN (B), each containing ammonium acetate (5 mM), from 15 to 100% B over 7 min, held at 100% B for 4 min, and re-equilibrated for 4 min. MS parameters were curtain gas 20 psi, capillary voltage 5.5 kV, temperature 450 °C, GS1/GS2 at 50 psi, CAD −3 arbitrary units, DP 70 V, EP 10 V, CE 70 eV, CXP 15 V. Analysis was by selected reaction monitoring (SRM) with 100 ms dwell times for each transition with Q1 ions selected at unit resolution for m/z 842.5, 844.5, 846.5, and 848.5 for AZA1; m/z 828.5, 830.5, 832.5, and 834.5 for AZA3; and m/z 856.5 for AZA2, all with the same Q3 ions measured using the low-resolution setting at m/z 362.2.
7
Quantification of Tea Compounds
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Gallic acid, catechins and alkaloids contents were determined following the method in our laboratory using a HPLC system [16 (link)]. HPLC analysis was carried out using an Agilent 1260 LC system (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a diode array detector (detection wavelength, 280 nm) coupled to an Inertisil ODS-3 (5 μm, 4.6 mm × 250 mm; GL-science Inc., Tokyo, Japan). External standard method was used to conduct quantification and the contents were shown as mg/g tea.
8
Analytical Characterization of Organic Compounds
9
LC-MS/MS Analysis of Metabolites
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Two kinds of mass spectrometry were used in this study. One was Agilent 1260 LC system coupled with an Agilent 6540 quadrupole/time of flight mass spectrometry (LC-Q-TOF/MS, Agilent Technologies, Palo Alto, CA, United States). Chromatographic separation was carried on a Hedera ODS-2 C18 analytical column (150 mm × 2.1 mm, 5 μm; Hanbon Science and Technology, Huai’an, China) with a security Guard-C18 column (4 mm × 2.0 mm, 5 μm; Phenomenex, Torrance, CA, United States). The flow rate was 0.3 mL/min and the mobile phase of methanol (solvent A) and water containing 0.1% acetic acid (solvent B) in a linearly gradient program was conducted. The gradient program was as the following condition: 0–3 min, 95% B; 3–15 min, 95–30% B; 15–20 min, 30–10% B; 20–25 min, 10% B; 25–26 min, 10–95% B; 26–32 min, 95% B. The column temperature was maintained at 55°C. The injection volume was 10 μL. The mass spectrometry was equipped with an electrospray ionization source and operated in the positive mode with the full scan range of m/z 100–m/z 1300. Besides, auto MS/MS and targeted MS/MS modes were chosen for obtaining more fragmented ion information of analytes. Drying gas temperature of 350°C with N2 gas flow at 10 L/min, nebulizer pressure of 45 psi, capillary voltage of 4000 V, fragmentor of 135 V and collision energy of 25 eV were set.
10
LC-QTOF Analysis of MsPDE Products
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The products of MsPDE were identified by LC/Q-TOF analysis, which was performed on Agilent 1260 LC system (Agilent Technologies, USA) coupled to an ultra high definition quadrupole time-of-flight mass spectrometer Model 6540 (Agilent Technologies, USA) equipped with a dual source electrospray ionization ion source.
The reaction products were separated on Agilent C18 reverse-phase column, with the binary mobile phase composed of 2% methanol and 98% water (containing 0.2% ammonium acetate and 0.1% acetic acid) being set at a constant flow rate of 300 μL/min and column temperature of 30 °C.
Q-TOF parameters were as follows: ionization mode, positive mode; capillary voltage: 4,000 V; drying gas: 9 L/min; nebulizer pressure: 40 psig; gas temperature: 350 °C; skimmer voltage: 65 V; octopole RF Peak voltage: 750 V, fragmentor voltage: 150 V. LC/MS accurate mass spectra were recorded across a range of 100-1000 m/z at the MS scan rate 1.5 spectra/s. Accurate mass measurements of each peak from the total ion chromatograms were obtained by an automated calibrant delivery system using a low flow of a calibrating solution (calibrant solution A, Agilent Technologies), which contains the internal reference masses purine (C5H4N4) at m/z 121.0509 and HP-921 [hexakis-(1H, 1H, 3H-tetrafluoro-pentoxy) phosphazene] (C18H18O6N3P3F24) at m/z 922.0098.
The reaction products were separated on Agilent C18 reverse-phase column, with the binary mobile phase composed of 2% methanol and 98% water (containing 0.2% ammonium acetate and 0.1% acetic acid) being set at a constant flow rate of 300 μL/min and column temperature of 30 °C.
Q-TOF parameters were as follows: ionization mode, positive mode; capillary voltage: 4,000 V; drying gas: 9 L/min; nebulizer pressure: 40 psig; gas temperature: 350 °C; skimmer voltage: 65 V; octopole RF Peak voltage: 750 V, fragmentor voltage: 150 V. LC/MS accurate mass spectra were recorded across a range of 100-1000 m/z at the MS scan rate 1.5 spectra/s. Accurate mass measurements of each peak from the total ion chromatograms were obtained by an automated calibrant delivery system using a low flow of a calibrating solution (calibrant solution A, Agilent Technologies), which contains the internal reference masses purine (C5H4N4) at m/z 121.0509 and HP-921 [hexakis-(1H, 1H, 3H-tetrafluoro-pentoxy) phosphazene] (C18H18O6N3P3F24) at m/z 922.0098.
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