For analysis of isolated compounds, an HPLC ekspert ultraLC 100 coupled with a TripleTOF 5600 mass spectrometer (AB SCIEX, Framingham, MA, USA) was used. For chromatographic separation, a Shim-pack XR-ODS C18 column (5 cm × 2.0 mm, 2.2 µm particle size) (Shimadzu, Kyoto, Japan) was used, coupled with a pre-column with the same material. The mobile phases were water and methanol (both with 0.1% of formic acid). The elution method initiated with 5% of methanol was kept isocratic for 1.0 min, which was then increased to 95% over 10 min. Four minutes were added to the method to wash and stabilize the column. The injection volume was 2 µL, and the column temperature was 40 °C. The positive ionization mode was employed and the ionization source parameters were CUR 22 (Curtain Gas), source temperature 450 °C, ionization voltage (IS) 5500 V, Gas1 (nebulization gas) 45, Gas2 (turbo heaters gas) 45 and DP 100 (declustering potential).
Shim pack xr ods c18 column
Manufactured by Shimadzu
Sourced in Japan
The Shim-pack XR-ODS C18 column is a high-performance liquid chromatography (HPLC) column. It features a reversed-phase packing material consisting of porous silica particles bonded with octadecylsilane (C18) ligands. The column is designed for the separation and analysis of a wide range of compounds, including polar and non-polar molecules.
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Lab products found in correlation
Ekspert ultralc 100, by AB Sciex (1 mentions)
Tripletof 5600 mass spectrometer, by AB Sciex (1 mentions)
Compass hystar, by Bruker (1 mentions)
Solarix 7.0t ft icr ms system, by Bruker (1 mentions)
Nexera x2, by Shimadzu (1 mentions)
Lcms 8040, by Shimadzu (1 mentions)
Lc 30ad pump, by Shimadzu (1 mentions)
1100 series hplc system, by Agilent Technologies (1 mentions)
Spectramax m5, by Molecular Devices (1 mentions)
4 protocols using shim pack xr ods c18 column
1
HPLC-MS/MS Analysis of Isolated Compounds
2
Metabolic Profiling by UHPLC-FT-ICR MS
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All analysis were performed on a UHPLC-FT-ICR MS instrument, an Agilent 1260 system coupled with a Bruker Solarix 7.0 T FT-ICR MS system (Bruker, Germany) equipped with an electrospray ionization source (ESI). Chromatographic separation was performed on a Shim-pack XR-ODS C18 column (75 mm × 3.0 mm, 2.2 μm) (SHIMADZU, Japan), the column temperature was maintained at 35 °C, the flow rate was set at 0.50 ml min−1. The mobile phase, consisting of 0.1% formic acid water (A) and acetonitrile (B), was delivered using a liner gradient program as follows: 10–20% (B) in 0–13 min, 20–35% (B) in 13–18 min, 35–50% (B) in 18–20 min, 50–50% (B) in 20–30 min. The injection volume was 5 μl.
For MS detection, the instrument was operated in negative ion mode, and full-scan mass rage was 100–1000 Da. The optimal conditions were as follows: a nebulizer gas pressure of 4.0 bar, a dry gas flow rate of 8.0 L min−1, a capillary voltage of −3.5 kV, an end plate off set of −500 V, and a transfer capillary temperature of 250 °C. While in MS/MS experiments, the collision energy was initially set at 20 V of the preferred ions and then adjusted according to the fragments. FT MS control and Bruker Compass-Hystar (Bruker, Germany) were used to control the equipment and for data acquisition and analysis, respectively.
For MS detection, the instrument was operated in negative ion mode, and full-scan mass rage was 100–1000 Da. The optimal conditions were as follows: a nebulizer gas pressure of 4.0 bar, a dry gas flow rate of 8.0 L min−1, a capillary voltage of −3.5 kV, an end plate off set of −500 V, and a transfer capillary temperature of 250 °C. While in MS/MS experiments, the collision energy was initially set at 20 V of the preferred ions and then adjusted according to the fragments. FT MS control and Bruker Compass-Hystar (Bruker, Germany) were used to control the equipment and for data acquisition and analysis, respectively.
3
Optimized UHPLC-MS/MS for Glycyrrhizin
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Chromatographic development was performed on Shimadzu NEXERA-X2, UHPLC (Ultra High Performance Liquid Chromatograph) system with LC-30AD pumps, SIL-30A autosampler and CTO-20AC as column oven. The data processor used LabSolutions software. Optimization MRM transitions for glycyrrhizin was done on Shimadzu LCMS-8040 model (Triple Quadrupole Mass Spectrometer). Analysis was performed on shimadzu, shim-pack XR-ODS, C18 column (L 75 mm × 3.0 mm; 2.2 μm). Mobile phase-A as 20 mM Ammonium acetate in water and mobile phase-B as acetonitrile was used after being filtered through a 0.2 m Millipore membrane filter and degassed by sonication. The flow rate was maintained at 0.3 mL min−1 and the injection volume is 5 μL. Gradient method was employed for chromatographic separation of Glycyrrhizin from the matrix. The gradient program was 0.05 min–25 % B, 1.0 min–60 % B, 3.0 min–90 % B, 5.0 min–90 % B and 8.0 min–25 % B. The temperature in the column oven was set to 40 °C.
Analysis was performed using APCI (Atmospheric Pressure Chemical Ionization) interface at positive mode with a capillary voltage of 4 V. The following MS parameters were used in the analysis: Nebulizing Gas flow: 2 L min−1, Drying Gas flow: 15 L min−1, Interface temperature: 350 °C, DL (desolvation Line) temperature: 200 °C, and Heating Block: 400 °C.
Analysis was performed using APCI (Atmospheric Pressure Chemical Ionization) interface at positive mode with a capillary voltage of 4 V. The following MS parameters were used in the analysis: Nebulizing Gas flow: 2 L min−1, Drying Gas flow: 15 L min−1, Interface temperature: 350 °C, DL (desolvation Line) temperature: 200 °C, and Heating Block: 400 °C.
4
Quantification of Cardiac Glycosides by LC-MS/MS
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24R-epimer, 24S-epimer and digoxin were quantified by LC-MS/MS. The system consisted of an 1100 series HPLC system (Agilent Technologies, Waldbronn, USA) and a TSQ Quantum Access tandem mass spectrometer (Thermo Electron Corporation, San Josem, CA, USA) with an electrospray ionization source. Selective reaction monitoring (SRM) in the positive ionization mode was applied to quantity. The analytical columns were a Shim-pack XR-ODS C18 column (50 mm×2.1 mm i.d., 2.2 µm, Shimadzu, Japan). The rat plasma samples, cell lysates and buffer solutions were all prepared by liquid–liquid extraction. Detailed extraction solvents, LC conditions and mass parameters were shown in Table 1 .
Rhodamine 123 and phenol red in cell lysates or intestinal perfusion buffers were quantified by spectroscopy using enzyme-labeled instrument (SpectraMax M5,Molecular Device, USA). An aliquot of 200 µl of sample was added in 96-well plates, the excitation and emission maxima for rhodamine 123 were 485 and 535 nm, respectively. The detection wavelength of phenol red was 562 nm.
Rhodamine 123 and phenol red in cell lysates or intestinal perfusion buffers were quantified by spectroscopy using enzyme-labeled instrument (SpectraMax M5,Molecular Device, USA). An aliquot of 200 µl of sample was added in 96-well plates, the excitation and emission maxima for rhodamine 123 were 485 and 535 nm, respectively. The detection wavelength of phenol red was 562 nm.
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