The methylglyoxal trapping assay followed a published method with some modifications [5 (link),8 (link)]. Methylglyoxal (10 mM), o-phenylenediamine (50 mM) were freshly prepared in phosphate buffer (50 mM, pH 7.4). The final concentration of test samples (the five fractions, compounds 1 –3 and kaempferol) and aminoguanidine was 5 mg/mL. Three mL methylglyoxal were incubated with 0.75 mL test samples or phosphate buffer in pH 7.4 at 37 °C water bath for 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12 and 24 h. After incubation, 200 μL of o-phenylenediamine were added with 200 μL of each test solution. Before HPLC analysis, the mixtures were kept in dark at room temperature for 1 h. The remaining methylglyoxal was detected on an Thermo Dionex Ultimate 3000 HPLC system (Thermo Fisher Scientific Inc., Massachusetts, USA) equipped with a diode array detector, and a Thermo Syncronis-C18 column (250 mm × 4.6 mm i.d., 5 μm; Thermo Fisher Scientific Inc., Massachusetts, USA). Mobile phases were composed of 0.1% formic acid in water (A) and methanol (B). The elution started at 50% B and then increased to 55% in 10 min, and then increased to 100% in 11 min and lasted for 5 min, then returned to 50% in 16.5 min and lasted for 5 min. The flow rate was 1 mL/min and the injection volume was 20 μL. Detection wavelength was monitored at 315 nm.
Thermo syncronis c18 column
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Thermo Syncronis C18 column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of compounds. The column features a C18 stationary phase, which is a common choice for reversed-phase chromatography, and is suitable for use in various analytical applications.
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Lab products found in correlation
Alliance 2695 system, by Waters Corporation (2 mentions)
Dionex ultimate 3000 uhplc system, by Thermo Fisher Scientific (2 mentions)
Thermo dionex ultimate 3000 hplc system, by Thermo Fisher Scientific (1 mentions)
Dionex ultimate 3000 ultra high performance liquid chromatography system, by Thermo Fisher Scientific (1 mentions)
Acquity uplc liquid chromatography system, by Waters Corporation (1 mentions)
Xevo tq s ms ms system, by Waters Corporation (1 mentions)
Agilent 1100 series high, by Agilent Technologies (1 mentions)
8 protocols using thermo syncronis c18 column
1
Methylglyoxal Trapping Assay Protocol
2
High-Performance Liquid Chromatography Analysis
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Liquid Chromatography (LC) was conducted using the Dionex UltiMate 3000 Ultra High-performance Liquid Chromatography system (Thermo Fisher Scientific, Massachusetts, USA) equipped with a Thermo Syncronis C18 column (2.1 mm × 100 mm × 1.7 µm) (Thermo Fisher Scientific). During the analysis, the temperature was retained at 55 °C at a flow rate of 450 µL/min. Solvent A (water mixed with 0.1% formic acid) and solvent B (acetonitrile mixed with 0.1% of formic acid) were used as the mobile phases. Beginning at 0.5% of solvent B, the gradient elution program was set for 1 min, followed by 15 min from 0.5% to 99.5% of solvent B before being maintained for 4 min, and returned to 0.5% of solvent B in 2 min. The injection volume was set at 2 µL.
3
Extraction and Analysis of Green Tea Catechins
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Fresh green tea (Camellia sinensis) leaves were collected in the spring from the Osulloc Tea Garden in Jeju, Korea and dried. Dried C. sinensis leaves were subjected to extraction twice with 50% aqueous ethanol and incubation at 100°C (1.2 atm) under aqueous conditions for 5 h to obtain GCG-GTE. The catechin contents in GCG-GTE were measured and analyzed using high-performance liquid chromatography (HPLC) with a photodiode array (PDA) detector (Alliance 2695 system, Waters) and a Thermo Syncronis C18 column (250 × 4.6 mm, I.D., 5 μm; Thermo Fisher Scientific Inc.) GCG-GTE contained almost the same amount of epicatechins and epicatechin epimers (Table 1 ).
4
HPLC-PDA Analysis of HTP-GTE
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HTP-GTE was analyzed by HPLC-PDA (Alliance 2695 system, Waters, USA) using a ThermoSyncronis C18 column (250 × 4.6 mm, I.D., 5 μm; Thermo Fisher Scientific Inc.). The mobile phases were 0.1% acetic acid in water for solvent A and acetonitrile for solvent B. The gradient elution was 90% A + 10% B at 0–10 min, 85% A + 15% B at 10–30 min, 80% A + 20% B at 30–53 min, 5% A + 95% B at 53–55 min, 90% A + 10% B at 55–60 min with a flow rate of 1.0 mL/min. The sample volume for injection was 10 μl, and the measurement was carried at the UV wavelength of 280 nm.
5
UPLC-MS/MS Quantification of R-/S-HFBA
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An ACQUITY UPLC™ liquid chromatography system equipped with a quaternary pump, automatic injector, oven and a Xevo TQS MS/MS system (Waters Corporation, Milford, MA, USA) was used for excretion analyses. Chromatographic separation was performed on a Thermo Syncronis C18 column (50 mm × 2.1 mm, 1.7 μm; Thermo, USA). The analytes were eluted using a gradient method at a flow rate of 0.4 mL min−1. Mobile phase A was 0.1% formic acid in water and mobile phase B was acetonitrile according to the following gradient: 0–1.5 min, 15–46% B; 1.5–2.5 min, 46% B; 2.5–2.6 min, 46–15% B; 2.6–3.5 min, 15% B. The injection volume was 2 μL. The column and automatic injector temperature were set at 35 °C and 4 °C, respectively.
The mass spectrometer was operated in positive electrospray mode using multiple reaction monitoring (MRM). The desolvation temperature and source temperature were 350 °C and 150 °C, respectively. The capillary voltage was 3.0 kV. The desolvation gas flow rate and the cone gas flow rate were 700 L h−1 and 150 L h−1, respectively. The ion transitions (m/z) were 278.0 > 187.9 for R-/S-HFBA, 151.9 > 109.9 for IS. The collision energy were 18 eV for R-/S-HFBA and 14 eV for IS, respectively. The cone voltage were 21 V for R-/S-HFBA and 22 V for IS, respectively. The product ion spectra for R-/S-HFBA and IS were shown in our previous report.23 (link)
The mass spectrometer was operated in positive electrospray mode using multiple reaction monitoring (MRM). The desolvation temperature and source temperature were 350 °C and 150 °C, respectively. The capillary voltage was 3.0 kV. The desolvation gas flow rate and the cone gas flow rate were 700 L h−1 and 150 L h−1, respectively. The ion transitions (m/z) were 278.0 > 187.9 for R-/S-HFBA, 151.9 > 109.9 for IS. The collision energy were 18 eV for R-/S-HFBA and 14 eV for IS, respectively. The cone voltage were 21 V for R-/S-HFBA and 22 V for IS, respectively. The product ion spectra for R-/S-HFBA and IS were shown in our previous report.23 (link)
6
UHPLC-Based Analytical Method for Identification
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Liquid chromatography (LC) was performed using a Dionex UltiMate 3000 UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA) coupled to a Thermo Syncronis C18 column (2.1 mm × 100 mm × 1.7 μm; Thermo Fisher Scientific, Waltham, MA, USA), which was maintained at 55 °C and a flow rate of 450 µL/min during analysis. The mobile phases consisted of solvent A (water added with 0.1% formic acid) and solvent B (acetonitrile added with 0.1% formic acid). The gradient elution program was started at 0.5% of solvent B for 1 min, then from 0.5% to 99.5% of solvent B for 15 min and held for 4 min. The columns were then conditioned for 2 min before the next injection. The injection volume was set to 2 µL.
7
HPLC Analysis of Antifungal Compounds
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The active fraction collected from SPE separation was analyzed further by a model of Agilent 1100 series high-performance liquid chromatography (Agilent Technologies). Compounds were separated in a Thermo Syncronis C-18 column (5 μm, 250 × 4.6 mm, Thermo Fisher Scientific) using a gradient solvent system composed of water-methanol at a flow rate of 1 mL/min as the mobile phase. The concentrations of methanol were programmed as follows: 5–42% at 0–5 min, 42–44% at 5–10 min, 44–45% at 10–25 min, 45–95% at 25–30 min, and hold in 95% at 30–40 min. Compounds were detected by a UV detector with an optical absorbance at 254 nm. Each peak was collected, dried, and tested for antifungal activity.
8
UHPLC Method for Compound Analysis
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Liquid chromatography (LC) was performed using the Dionex UltiMate 3000 UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA) coupled with a Thermo Syncronis C18 column (2.1 mm × 100 mm × 1.7 μm; Thermo Fisher Scientific, Waltham, MA, USA), which was maintained at 55 °C at a flow rate of 450 µL/min during analysis. The mobile phases were composed of solvent A (water added with 0.1% formic acid) and solvent B (acetonitrile added with 0.1% of formic acid). The gradient elution program was initiated at 0.5% of solvent B for 1 min, then from 0.5% to 99.5% of solvent B for 15 min and maintained for 4 min. Later, the columns were conditioned as initial for 2 min before the next injection. The injection volume was set at 2 µL.
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