Chromatographic separation was performed using the Waters XSelect HSS T3 C18 column (2.1 mm × 50 mm, 3.5 μm). Mobile phase A consisted of 0.1% formic acid (v/v) in water, and mobile phase B was methanol. Gradient elution was performed at a constant flow rate of 0.4 ml/min with an injection volume of 5 μl. The mass spectrometer was operated in positive electrospray ionization (ESI) mode. The temperature of the ion source was 250°C, and the ion spray voltage was 3,500 V. Nitrogen was used as the nebulizer gas at a pressure of 45 psi. The sheath gas temperature and flow were 350°C and 11 l/min, respectively. The fragmentation voltage and collision energies were 135 V and 30 eV for TGC and IS, respectively. Quantification was performed using multiple reaction monitoring (MRM) at m/z 586.3→m/z 513.1 for TGC, and m/z 595.2→m/z 514.2 for IS.
Xselect hss t3 c18 column
Manufactured by Waters Corporation
Sourced in United States
The XSelect HSS T3 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 and a superficially porous particle structure, which provides efficient and high-resolution separations.
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Lab products found in correlation
Xcalibur, by Thermo Fisher Scientific (1 mentions)
Vanquish lc system, by Thermo Fisher Scientific (1 mentions)
Tracefinder, by Thermo Fisher Scientific (1 mentions)
Q exactive focus orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Dionex ultimate 3000, by Thermo Fisher Scientific (1 mentions)
Waters alliance e2695 hplc system, by Waters Corporation (1 mentions)
Empower 3, by Waters Corporation (1 mentions)
Hplc system, by Waters Corporation (1 mentions)
5 protocols using xselect hss t3 c18 column
1
HPLC-MS/MS Quantification of Trogocitinib
2
Targeted LC-MS/MS Analysis of Higenamine
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Analysis was conducted using a Vanquish LC system coupled to a Q‐Exactive Focus Orbitrap mass spectrometer (Thermo, Hemel Hempstead, UK). The LC was equipped with a Waters X‐Select HSS T3 C18 column (2.1 × 75 mm, 2.5 μm) using 0.1% formic acid in water (A) and 0.1% formic acid in methanol (B) as mobile phases. A gradient was utilised, with flow rate 0.48 ml/min, starting at 0% B for 1 min increasing to 10% B within 2.2 min, 60% within 4 min and increasing to 100% B within 6.5 min. The system was held at 100% B for 1 min prior to re‐equilibration at starting conditions.
The mass spectrometer was operated in positive ionisation mode with a HESI‐II (Heated Electro Spray Ionisation) probe using a vaporiser temperature of 420°C and ion transfer tube temperature of 320°C. The spray voltage was 2500 V. The mass spectrometer was operated in full scan data dependent Discovery (ddDiscMS/MS) mode to capture both MS and MS2 data concurrently. MS2 data were derived from the fragmentation of ions detected above a threshold. Subsequent analysis was performed utilising Parallel Reaction Monitoring (PRM) mode to ensure MS2 data were captured for both higenamine and the IS. Data were processed using Thermo XCalibur version 4.0.27.21 QualBrowser and Thermo TraceFinder version 4.1.
The mass spectrometer was operated in positive ionisation mode with a HESI‐II (Heated Electro Spray Ionisation) probe using a vaporiser temperature of 420°C and ion transfer tube temperature of 320°C. The spray voltage was 2500 V. The mass spectrometer was operated in full scan data dependent Discovery (ddDiscMS/MS) mode to capture both MS and MS2 data concurrently. MS2 data were derived from the fragmentation of ions detected above a threshold. Subsequent analysis was performed utilising Parallel Reaction Monitoring (PRM) mode to ensure MS2 data were captured for both higenamine and the IS. Data were processed using Thermo XCalibur version 4.0.27.21 QualBrowser and Thermo TraceFinder version 4.1.
3
Quantification of Quercetin in SCH Extracts
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The quercetin standard was dissolved in methanol, generating concentrations of 0.1, 0.05, 0.02, 0.01, and 0.005 mg/ml, which were used to prepare the standard curves for quercetin quantification in the tested sample. Aqueous extracts of SCH (100 mg) were extracted via ultrasonication in methanol (1 ml) for 20 min. The extracted solutions were subjected to a 0.22-μm filter membrane, yielding a clear solution for high-performance liquid chromatography (HPLC) analysis. Analytical HPLC experiments were conducted on a Dionex UltiMate 3000 instrument (Thermo Scientific, Waltham, MA, USA) equipped with a diode array detector and a Waters XSelect HSS T3 C18 column (250 × 4.6 mm, 5 μm). The chromatographic conditions for HPLC analysis were as follows: acetonitrile-H2O (0.1% H3PO4) (30:70, v/v), flow rate of 1 ml per min, column temperature of 30°C, run time of 20 min, and a detected wavelength of 360 nm.
4
HPLC Analysis of 3,5-DCA Metabolites
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Samples (75 µL) of media and lysed IKC were analyzed on a Waters Alliance e2695 HPLC system (Waters Co., Milford, MA, USA), utilizing a Waters 2489 variable UV/Vis detector at 254 nM. Chromatograms were collected and integrated using Empower 3 software (Waters Co., Milford, MA, USA). A Waters X Select HSS T3 C18 column (3.5 µm; 4.6 × 150 mm2) fitted with a Waters XSelect HSS T3 VanGuard Cartridge (3.5 µm; 3.9 × 5 mm2) was used for separation of compounds. The mobile phase consisted of 50:50 methanol:water with a flow rate of 0.75 mL/min. Standard curves, limits of detection, and extraction coefficients were determined for 3,5-DCA and putative metabolites. Extraction coefficients were determined by comparing the integration of a 1.0 mM 3,5-DCA or a metabolite sample that had undergone sample processing (i.e., the addition of methanol and centrifugation) to that of a unprocessed 1.0 mM 3,5-DCA or metabolite sample.
5
HPLC Analysis of Chromatographic Compounds
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Chromatographic analysis was performed with a HPLC system (Waters Co., Milford, MA, USA) and a photodiode array detector. For the HPLC analysis of SDE, the prim-O-glucosylcimifugin standard was purchased from the Korea Promotion Institute for Traditional Medicine Industry (Gyeongsan, Korea), and sec-O-glucosylhamaudol and 4′-O-β-D-glucosyl-5-O-methylvisamminol were isolated within our laboratory and identified by spectral analyses, primarily by NMR and MS.
SDE samples (0.1 mg) were dissolved in 70% ethanol (10 mL). Chromatographic separation was performed with an XSelect HSS T3 C18 column (4.6 × 250 mm, 5 μm, Waters Co., Milford, MA, USA). The mobile phase consisted of acetonitrile (A) and 0.1% acetic acid in water (B) at a flow-rate of 1.0 mL/min. A multistep gradient program was used as follows: 5% A (0 min), 5–20% A (0–10 min), 20% A (10–23 min), and 20–65% A (23–40 min). The detection wavelength was scanned at 210–400 nm and recorded at 254 nm. The injection volume was 10.0 μL. Standard solutions for the determination of three chromones were prepared at a final concentration of 7.781 mg/mL (prim-O-glucosylcimifugin), 31.125 mg/mL (4′-O-β-D-glucosyl-5-O-methylvisamminol), and 31.125 mg/mL (sec-O-glucosylhamaudol) in methanol and kept at 4°C.
SDE samples (0.1 mg) were dissolved in 70% ethanol (10 mL). Chromatographic separation was performed with an XSelect HSS T3 C18 column (4.6 × 250 mm, 5 μm, Waters Co., Milford, MA, USA). The mobile phase consisted of acetonitrile (A) and 0.1% acetic acid in water (B) at a flow-rate of 1.0 mL/min. A multistep gradient program was used as follows: 5% A (0 min), 5–20% A (0–10 min), 20% A (10–23 min), and 20–65% A (23–40 min). The detection wavelength was scanned at 210–400 nm and recorded at 254 nm. The injection volume was 10.0 μL. Standard solutions for the determination of three chromones were prepared at a final concentration of 7.781 mg/mL (prim-O-glucosylcimifugin), 31.125 mg/mL (4′-O-β-D-glucosyl-5-O-methylvisamminol), and 31.125 mg/mL (sec-O-glucosylhamaudol) in methanol and kept at 4°C.
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