The chromatographic separation of compounds was performed on a Shimadzu Prominence LC-20A System (Shimadzu, Kyoto, Japan) equipped with a degasser (DGU-20A5), a binary gradient pump (LC-20AB), and a diode array detection system (SPD-M20A, Shimadzu). The column used was a 5-μm Hypersil ODS C18 column (4.6 mm × 250 mm, i.d.), which was operated at a temperature of 30 °C. Methanol (A)-H2O (B) was used as the mobile phase. The LC gradient program was set as (Tmin/A:B; T0/40:60; T8.0/40:60; T10/50:50; T20/40:60; T26/40:60). The flow rate was 1.0 mL·min−1. The injection volume was 20 μL, and the wavelengths were set at 210 nm and 254 nm.
Prominence lc 20a system
Manufactured by Shimadzu
Sourced in Japan, United States
The Prominence LC-20A system is a high-performance liquid chromatography (HPLC) instrument manufactured by Shimadzu. It is designed to perform liquid chromatographic analysis and separation of various chemical compounds. The system includes a solvent delivery unit, an autosampler, a column oven, and a variety of detectors to monitor the separated analytes.
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Lab products found in correlation
Gemini c18 column, by Phenomenex (5 mentions)
Superdex 200 increase 10 300 gl column, by Cytiva (4 mentions)
Labsolution version 5, by Shimadzu (3 mentions)
Frc 10a, by Shimadzu (3 mentions)
Superdex 200 increase 10 300 gl column, by GE Healthcare (3 mentions)
Spd m20a, by Shimadzu (2 mentions)
Lc solution software, by Shimadzu (2 mentions)
Labsolution software, by Shimadzu (2 mentions)
Milli q water purification system, by Merck Group (2 mentions)
Lc 20ab, by Shimadzu (1 mentions)
Dgu 20a5, by Shimadzu (1 mentions)
Lcsolution software version 1.24, by Shimadzu (1 mentions)
Lc 20at pump, by Shimadzu (1 mentions)
Sil 20ac auto, by Shimadzu (1 mentions)
Dgu 20a3, by Shimadzu (1 mentions)
0.2 μm membrane filter, by Pall Corporation (1 mentions)
Premier c18 column, by Shimadzu (1 mentions)
Sunfire c18 column, by Waters Corporation (1 mentions)
Api 4000 qtrap mass spectrometer, by AB Sciex (1 mentions)
Pack pro c18 column, by YMC (1 mentions)
33 protocols using prominence lc 20a system
1
Reverse-Phase HPLC Separation of Compounds
2
HPLC Analysis of Aromatic Amino Acids
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All samples were analyzed by Shimadzu Prominence LC-20A system (Shimadzu, Kyoto, Japan) equipped with a reversed phase column (Shimadzu Insert Sustain C18 column: 150 mm × 4.6 mm × 5 µm) and a photodiode array detector (DAD). Mobile phase for isocratic analysis: 70% water with 0.1% trifluoroacetate + 30% acetonitrile. Flow rate: 1 mL/min. Column temperature: 40 °C. Injection volume: 5 μL. Retention time: L-tyrosine 1.7 min; L-phenylalanine 2.1 min; 4HBA 2.9 min; benzoic acid 6.1 min. Authentic standards were used to plot the standard curves and the product concentrations were calculated accordingly.
3
Simultaneous Determination of Bioactive Compounds
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Chromatographic analysis for simultaneous determination was performed on a Shimadzu Prominence LC-20A system (Shimadzu Co., Kyoto, Japan), comprising a solvent delivery unit, online degasser, column oven, autosampler, and photodiode array (PDA) detector. The data processor used LC Solution Software (version 1.24; Shimadzu Co.). The nine constituents were separated on a Phenomenex Gemini C18 column (250 mm × 4.6 mm, 5 μm; Phenomenex, Torrance, CA) and maintained at 40°C. The mobile phases comprised of 0.1% (v/v) formic acid in water (A) and acetonitrile (B). The gradient flow was as follows: 10–60% B for 0–30 min, 60–100% B for 30–40 min, 100% B for 40–45 min, 100–10% B for 45–50 min, and 100% B for 50–60 min. The flow rate was 0.6 mL/min, and the injection volume was 10 μL. The wavelength of the PDA was 190–400 nm, and the detected wavelengths for quantitative analysis were monitored at 240 nm (neomangiferin, mangiferin, geniposide, and sweroside), 280 nm (pinoresinol), and 325 nm (chlorogenic acid and caffeic acid). All calibration curves were obtained by assessment of the peak areas from standard solutions in the following concentration ranges: Neomangiferin and chlorogenic acid, 1.56–50.00 μg/mL; mangiferin and geniposide, 6.25–200.00 μg/mL; sweroside, 3.13–100.00 μg/mL; and caffeic acid and pinoresinol, 0.63–20.00 μg/mL.
4
Purification of Hydrolyzed Peptides Using Preparative HPLC
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Hydrolyzed peptides, using Alcalase®, were purified using an Xbridge BEH preparative C18 5 μm OBD 19 × 250 mm (Waters) connected to a Shimadzu Prominence LC-20A system, including a CBM-20A controller, two LC-20 AP preparative pumps and a DGU-20A3R online degasser. An SPD-20A UV with a preparative cell (0.5 mm) was used as a detector and was set at 214nm. An FRC-10A Shimadzu was employed as an auto collector. Data acquisition was performed by the LabSolution version 5.53 software (Shimadzu, Kyoto, Japan).
The sample was eluted with a flow rate of 17 mL min−1 using ddH2O/TFA (99.9/0.1, v/v) as phase A and MeOH/TFA (99.9/0.1, v/v) as phase B. The gradient started with 1 min of 25% phase B, and then increased to 50% in 19 min; finally, B was increased to 95% in 1 min and maintained constant for 5 min. The column was re-equilibrated for 6 min at 25% B. Ten fractions were collected every 3 min (except for fraction 1 and 12, as shown inFigure 2 ). Collected peptide fractions (F1–F10) were subjected to bioactivity tests to identify the most active ones.
The sample was eluted with a flow rate of 17 mL min−1 using ddH2O/TFA (99.9/0.1, v/v) as phase A and MeOH/TFA (99.9/0.1, v/v) as phase B. The gradient started with 1 min of 25% phase B, and then increased to 50% in 19 min; finally, B was increased to 95% in 1 min and maintained constant for 5 min. The column was re-equilibrated for 6 min at 25% B. Ten fractions were collected every 3 min (except for fraction 1 and 12, as shown in
5
Separation and Quantification of 3,29-dibenzoyl-rarounitriol
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The analysis was performed using a Shimadzu Prominence LC–20A system (Shimadzu, Kyoto, Japan) consisting of a solvent delivery unit, an on-line degasser, a column oven, an autosampler, and a photodiode array (PDA) detector, was employed. The data processor used LCsolution software (version 1.24; Shimadzu, Kyoto, Japan). The 3,29-dibenzoyl-rarounitriol was separated on a Phenomenex Gemini C18 column (250 × 4.6 mm; particle size 5 μm, Phenomenex, Torrance, CA) and maintained at 40°C. The mobile phases for chromatographic separation were carried out using isocratic elution (A : B = 5 : 95) of solvent A (distilled water) and solvent B (acetonitrile) for 30 min. The analysis was performed at a flow-rate of 1.0 mL/min using a detection wavelength of 230 nm. The injection volume was 10 μL.
6
HPLC Analysis of Biomarkers in SCWE Decoction
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The quantitative analysis of the eight biomarker compounds in SCWE decoction was performed using a Shimadzu Prominence LC-20A system (Kyoto, Japan) equipped with LC-20AT pumps, DGU-20A3 online degasser, CTO-20A column oven, SIL-20AC auto sample injector, and SPD-M20A photodiode array (PDA) detector. All chromatographic data were recorded and processed by LCsolutions software (Version 1.24 SP1). The separation of the eight analytes was achieved on a Phenomenx Gemini C18 column (250 mm × 4.6 mm, 5 μm, Torrance, CA, United States), and column temperature was maintained at 40°C. The mobile phase consisted of 1.0% (v/v) aqueous acetic acid (A) and 1.0% (v/v) acetic acid in acetonitrile (B), and gradient conditions were as follows: 15–65% B (0–35 min), 65–100% B (35–45 min), 100% B (45–50 min), and 100–15% B (50–55 min). The flowrate was 1.0 mL/min, and the injection volume was 10 μL. For quantitative analysis, lyophilized 200 mg of SCWE extract was dissolved in 20 mL of distilled water and extracted at room temperature for 20 min using an ultrasonicator (Branson 8510, Danbury, CT, United States) The solution was then filtered through a 0.2 μm membrane filter (PALL Life Sciences, Ann Arbor, MI, United States) before the HPLC injection.
7
Characterization of Fucosyltransferase Enzyme Kinetics
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The prepared reaction mixture of 1 μL of 10 mM GDP-fucose, 1 μL of 10 mM lacto-N-tetraose (LNT), 6 μL of purified enzyme His6-Tp2FT, 1 μL of 10 mM MgCl2, 1 μL of 10 mM Tris-HCl was incubated at same pH (8.0) and different temperatures (15℃, 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃) for 5 min, and at same temperature (37℃) and different pH (3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0) 37℃ for 5 min. The pH value of buffer was adjusted with citric acid (pH 3.0–4.0), MES (pH 5.0–6.0) and Tris-HCl (pH 7.0–9.0). The same mixture (except MgCl2) at pH 7.0 was incubated with different concentrations of MgCl2 (1 μL of 5 mM, 10 mM, and 20 mM each), 10 mM EDTA, and 1 μL of 10 mM Tris-HCl at 37℃ for 5 min. The samples were analyzed by a Shimadzu Prominence LC-20A system equipped with a membrane online degasser, a temperature control unit, and a fluorescence detector. A reverse-phase Premier C18 column (250 × 4.0 mm i.d., 5 μm, Shimadzu) was used. The mobile phase was acetonitrile/water (65:35), 1% formic acid at a flow rate of 0.2 mL/min at 40℃ (Zhong et al., 2022 ). Glycan-containing fractions were analyzed by mass spectrometry.
8
Phytochemical Analysis of FX by HPLC
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The phytochemical analysis of FX was performed using a Shimadzu Prominence LC–20A system (Kyoto, Japan) equipped with a photodiode array (PDA) detector. LC solution software (Version 1.24, SP1, Kyoto, Japan) was employed for the acquisition, processing, and conversion of chromatographic data. A Waters SunFire C18 column (250 × 4.6 mm, 5 μm, Milford, MA, United States) maintained at 40°C was used to separate the three marker components in the FX sample. The mobile phases consisted of 0.1% aqueous formic acid and 0.1% (v/v) formic acid in acetonitrile. The gradient elution of the mobile phase was as follows: 5–60% B for 0–40 min, 60% B for 40–45 min, and 60–5% B for 45–50 min. The flow-rate and injection volume were 1.0 mL/min and 10 μL, respectively.
9
LC-MS/MS Analysis of Folates, Hcy, AdoMet, AdoHcy
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Folates in tissue samples and erythrocytes were determined by means of LC-MS/MS (Finnigan Surveyor Plus high performance liquid chromatography (HPLC) System, Thermo Electron Corporation, Waltham, MA, USA; triple quadrupole TSQ quantum discovery mass spectrometer, Thermo Electron Corporation, Waltham, MA, USA). The vitamers were separated on a YMC Pack Pro C18 column (150 × 3 mm, 3 µm, YMC, Kyoto, Japan). The mobile phase for gradient elution consisted of 0.1% (v/v) aqueous formic acid (eluent A) and acetonitrile containing 0.1% (v/v) formic acid (eluent B) at a flow rate of 0.3 mL/min.
The system for Hcy, AdoMet, and AdoHcy measurement consisted of a Shimadzu Prominence LC-20A System (Shimadzu, Kyoto, Japan) and an API 4000 Q-Trap mass spectrometer (AB Sciex, Foster City, CA, USA). Analyte separation was carried out on a Phenomenex Gemini reversed phase column (110A 3u, 150 × 4.60 mm, Phenomenex, Aschaffenburg, Germany). The mobile phase for gradient elution consisted of 0.1% (v/v) aqueous formic acid (eluent A) and acetonitrile containing 0.1% (v/v) formic acid (eluent B) at a flow of 0.4 mL/min.
Gradients and source parameters of the LC-MS instrument for tissue, plasma samples [19 (link)], and erythrocytes [29 (link)] have been published earlier.
The system for Hcy, AdoMet, and AdoHcy measurement consisted of a Shimadzu Prominence LC-20A System (Shimadzu, Kyoto, Japan) and an API 4000 Q-Trap mass spectrometer (AB Sciex, Foster City, CA, USA). Analyte separation was carried out on a Phenomenex Gemini reversed phase column (110A 3u, 150 × 4.60 mm, Phenomenex, Aschaffenburg, Germany). The mobile phase for gradient elution consisted of 0.1% (v/v) aqueous formic acid (eluent A) and acetonitrile containing 0.1% (v/v) formic acid (eluent B) at a flow of 0.4 mL/min.
Gradients and source parameters of the LC-MS instrument for tissue, plasma samples [19 (link)], and erythrocytes [29 (link)] have been published earlier.
10
Quantitative Analysis of Gyejibokryeong-hwan Markers
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The chromatographic analysis for quantitative determination of the seven marker components in Gyejibokryeong-hwan was performed using a Shimadzu Prominence LC-20A system (Shimadzu Co., Kyoto, Japan) consisting of a solvent delivery unit, an on-line degasser, a column oven, a sample autoinjector, and a photodiode array (PDA) detector. The data were acquired and processed using LCsolution software (Version 1.24). The column used for separation of the seven constituents was a Phenomenex Gemini C18 column (250 × 4.6 mm, 5 μm, Torrance, CA, USA) maintained at 40 °C. The mobile phases consisted of water (A) and acetonitrile (B), both containing 1.0% (v/v) acetic acid. The gradient elution of the mobile phase was as follows: 10–60% B for 0–30 min, 60–100% B for 30–40 min, 100% B for 40–45 min, 100–10% B for 45–50 min, and 100% B for 50–60 min. The flow rate and injection volume were 1.0 mL/min and 10 μL, respectively. The detection wavelengths for quantitative analysis of the seven compounds were set according to the maximum absorption wavelengths of each reference compound (amygdalin, albiflorin, paeoniflorin for P. persica, coumarin, cinnamic acid, cinnamaldehyde for C.cassia, paeonol for P.suffruticosa)..
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