Method A; Column : Agilent XDB C18 column (9.4 × 250 mm, 5 μm). Mobile phase: A: water (0.1% TFA); B: acetonitrile (0.1% TFA). Gradient: 40 - 57% B in 7-min; flow rate of 4 mL/min. Method B; Column : Agilent XDB C18 column (4.6 × 150 mm, 5 μm). Mobile phase: A: water (0.1% TFA); B: acetonitrile (0.1% TFA). Gradient: 5 - 70% B in 15-min; flow rate of 1 mL/min. Method C; Column : Agilent Eclipse GF 250, 5 μm, 4.6 × 250 mm column, PBS buffer (pH 7.4), flowrate: 1 ml/min.
Xdb c18 column
Manufactured by Agilent Technologies
Sourced in United States, Germany
The XDB-C18 column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of analytes. It features a C18 stationary phase and is suitable for a variety of applications, including pharmaceutical, environmental, and food analysis.
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Lab products found in correlation
Prominence lc, by Shimadzu (4 mentions)
Sephadex lh 20, by GE Healthcare (2 mentions)
Agilent 1290 infinity uplc system, by Agilent Technologies (2 mentions)
1260 hplc system, by Agilent Technologies (2 mentions)
6410a triple quadrupole mass spectroscopy, by Agilent Technologies (2 mentions)
High performance liquid chromatography (hplc), by Agilent Technologies (2 mentions)
1200 hplc system, by Agilent Technologies (2 mentions)
1260 hplc, by Agilent Technologies (2 mentions)
4000 qtrap, by Shimadzu (2 mentions)
3200 qtrap, by Shimadzu (2 mentions)
N benzylbenzamide, by Merck Group (2 mentions)
Qtrap 4500, by AB Sciex (1 mentions)
Neoeriocitrin, by Merck Group (1 mentions)
Eriocitrin, by Merck Group (1 mentions)
Narirutin, by Merck Group (1 mentions)
Neohesperidin, by Merck Group (1 mentions)
Waters 2489, by Waters Corporation (1 mentions)
Waters e2695, by Waters Corporation (1 mentions)
Naringin, by Merck Group (1 mentions)
Agilent 1260, by Agilent Technologies (1 mentions)
85 protocols using xdb c18 column
1
Optimized HPLC Methods for Compound Analysis
2
Quantification of Vitamin D Metabolites
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Venous blood samples for vitamin D metabolites were collected at three time points (1 h before, and 1 h and 3 h after the exercise) into tubes containing a coagulation accelerator. The serum was separated using a laboratory centrifuge, aliquotted into 1.5-mL centrifuge tubes, and frozen at −80 °C until further analysis.
Before the analysis, serum proteins were precipitated and derivatized using a Cookson-type reagent (DAPTAD). Proteins were precipitated using acetonitrile. For 1,25(OH)2D3 determination, the sample preparation involved liquid–liquid extraction with ethyl acetate. Quantitative analyses were performed using liquid chromatography coupled with tandem mass spectrometry (Exion LC system coupled with QTRAP4500, Sciex, Framingham, MA, USA). Chromatographic separation was carried out using an XDB-C18 column (50 × 4.6 mm, 1.7 μm; Agilent, Santa Clara, CA, USA). Serum samples were analyzed in the positive ion mode, using electrospray ionization. The concentrations of the following vitamin D metabolites were determined: 25(OH)D3, 24,25(OH)2D3, 1,25(OH)2D3, 3-epi-25(OH)D3, and 25(OH)D2. The concentration range was 1–100 ng/mL for 25(OH)D3; 0.1–10 ng/mL for 25(OH)D2, 3-epi-25(OH)D3, and 24,25(OH)2D3; and 10–200 pg/mL for 1,25(OH)2D3. In addition, the ratios of 25(OH)D3 to 24,25(OH)2D3, and 25(OH)D3 to epi-25(OH)D3 were calculated.
Before the analysis, serum proteins were precipitated and derivatized using a Cookson-type reagent (DAPTAD). Proteins were precipitated using acetonitrile. For 1,25(OH)2D3 determination, the sample preparation involved liquid–liquid extraction with ethyl acetate. Quantitative analyses were performed using liquid chromatography coupled with tandem mass spectrometry (Exion LC system coupled with QTRAP4500, Sciex, Framingham, MA, USA). Chromatographic separation was carried out using an XDB-C18 column (50 × 4.6 mm, 1.7 μm; Agilent, Santa Clara, CA, USA). Serum samples were analyzed in the positive ion mode, using electrospray ionization. The concentrations of the following vitamin D metabolites were determined: 25(OH)D3, 24,25(OH)2D3, 1,25(OH)2D3, 3-epi-25(OH)D3, and 25(OH)D2. The concentration range was 1–100 ng/mL for 25(OH)D3; 0.1–10 ng/mL for 25(OH)D2, 3-epi-25(OH)D3, and 24,25(OH)2D3; and 10–200 pg/mL for 1,25(OH)2D3. In addition, the ratios of 25(OH)D3 to 24,25(OH)2D3, and 25(OH)D3 to epi-25(OH)D3 were calculated.
3
Flavanone Composition Analysis in Juice
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The flavanone composition was measured according to Huang et al. [29 (link)] with some modifications. Briefly, juice was diluted 2.33 times with methanol and flavonoid standards (eriocitrin, neoeriocitrin, narirutin, naringin, neohesperidin from Sigma-Aldrich (St. Louis, MO, USA)) were dissolved in methanol and sufficiently mixed. After filtration through a PTFE membrane with φ = 13 mm and pore size of 0.45 mm, 10 μL of samples (or standard solutions) were injected into the HPLC system (Waters e2695, Waters, Milford, MA, USA) coupled with a UV/vis detector (Waters 2489, Waters, Milford, MA, USA). The XDB-C18 column (250 × 4.6 mm, Agilent, Santa Clara, CA, USA) was kept at 25 °C with a flow rate of 0.7 mL/min. Solution A (0.1% formic acid) and solution B (methanol) were used as the mobile phase with the gradient elution as follows: 0–20 min, 63–50% A; 20–25 min, 50–20% A; 25–30 min, 20–0% A; 30–35 min, 0% A; 35–40 min, 0–63% A; 40–42 min, 63% A. The detection was performed at 283 nm. The flavonoids were identified and quantified according to the standard curves (Figure S1 ).
4
Analytical Methods for Black Sesame Components
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The moisture content was measured using the method of direct drying described by GB 5009.3-2016 (China). Crude protein was determined using the Kjeldahl method described by GB 5009.5-2016 (China). Fat was detected by the Soxhlet extraction method described by GB 5009.6-2016 (China). Total ash was determined by the first method described by GB 5009.4-2016 (China). Each sample was set to have three parallels.
Melanin was detected with an ultraviolet photometer (Bluestar, Beijing Labtek Instrument Co., Ltd., Beijing, China) at 360 nm. Alcohol was used to extract black sesame pigments, and the extraction technology conditions were as follows: 50% alcohol (v/v), 1:50 ratio of black sesame seeds to solvent, pH 1.0, and extraction for 30 min. Each sample was set to have three parallels.
Sesamin and sesamolin were measured using a high-performance liquid chromatography with UV detector (Agilent1260, Agilent Technology Co., Ltd., Palo Alto, CA, USA) described by NY/T 1595-2008 (China). The chromatographic conditions of HPLC were Agilent XDB-C18 column (4.6 mm × 250 mm, 5 μm); column temperature 30 °C; mobile phase: CH3OH/H2O = 80/20; flow rate 0.8 mL/min; and wavelength 290 nm. Each sample was tested twice. The curves of sesamin and sesamolin standards were shown inSupplementary File S1: Figures S1 and S2 .
Melanin was detected with an ultraviolet photometer (Bluestar, Beijing Labtek Instrument Co., Ltd., Beijing, China) at 360 nm. Alcohol was used to extract black sesame pigments, and the extraction technology conditions were as follows: 50% alcohol (v/v), 1:50 ratio of black sesame seeds to solvent, pH 1.0, and extraction for 30 min. Each sample was set to have three parallels.
Sesamin and sesamolin were measured using a high-performance liquid chromatography with UV detector (Agilent1260, Agilent Technology Co., Ltd., Palo Alto, CA, USA) described by NY/T 1595-2008 (China). The chromatographic conditions of HPLC were Agilent XDB-C18 column (4.6 mm × 250 mm, 5 μm); column temperature 30 °C; mobile phase: CH3OH/H2O = 80/20; flow rate 0.8 mL/min; and wavelength 290 nm. Each sample was tested twice. The curves of sesamin and sesamolin standards were shown in
5
Quantifying Polyamines in Cancer Cells
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The amount of natural polyamines (spermine, spermidine, putrescine) was analyzed by high performance chromatography (HPLC) in both hepatocellular and colorectal carcinoma transfected with SSAT or siSSAT. Briefly, the cells were harvested in 200 μL of PBS, and 200 μL of hexanediamine (1.624 μg/ml) was added as an internal standard. After adding 200 μL of dansyl chloride (5 mg/ml), the mixtures were incubated at 50°C for 30 min, and then the reaction was terminated by the addition of 1 mL of ethyl acetate. The supernatant containing the polyamines was functionalized with dansyl chloride and purified with an organic filtration. 20 μL of the sample was then injected onto a XDB-C18 column (4.6 ×250 mm, Agilent Technologies), which was achieved excitation at 340 nm and measured emission at 515 nm by fluorescence detector. The solvent system was consisted with methanol and water, running at 65% (v/v) to 100% (v/v) methanol with in 25 min at a flow rate of 1 mL/min. The polyamine content of cells treatment with exogenous polyamines (putrescine 0.08 μM, spermidine 0.5 μM, spermine 1.0 μM) were aslo measured in the same way.
6
Quantitative Analysis of Herbal Compounds
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Justicidin B, chinensinaphthol methyl ether, and 6’-hydroxy justicidin B were weighed accurately, and 1 mg/ml control solution with methanol was prepared. The control solution was stored at 4°C for use. The supernatant that was treated by solid phase microextraction cartridge was collected and dried by nitrogen after centrifugation (12,000 r/min, 10 min). Before the liquid injection, 200 μl acetonitrile was used for redissolution and the supernatant was determined by HPLC-DAD-ESI-MS after filtration.
The chromatography analysis was performed on an Agilent XDB-C18 column (150 mm × 4.6 mm, 4 µm) at 25°C; the mobile phase was a mixture of aqueous solutions containing water (A) and acetonitrile (B). The gradient elution procedure was made as follows: 0–14 min, 26% B; 14–51 min, 26–38.5% B; 51–80 min, 38.5% B. The injection volume was 100 μl, and the flow rate was 1 ml/min. The wavelength of 190–690 nm was used for the detection. Mass spectrometry detection was set as follows: capillary temperature, 200°C; source voltage, 4.5 kV for the positive ion mode. The mass range was from 50 to 1,600. Blood absorption compounds were identified by accurate mass, MS/MS ion fragment pattern, and retention time of LC and then were validated by available standard.
The chromatography analysis was performed on an Agilent XDB-C18 column (150 mm × 4.6 mm, 4 µm) at 25°C; the mobile phase was a mixture of aqueous solutions containing water (A) and acetonitrile (B). The gradient elution procedure was made as follows: 0–14 min, 26% B; 14–51 min, 26–38.5% B; 51–80 min, 38.5% B. The injection volume was 100 μl, and the flow rate was 1 ml/min. The wavelength of 190–690 nm was used for the detection. Mass spectrometry detection was set as follows: capillary temperature, 200°C; source voltage, 4.5 kV for the positive ion mode. The mass range was from 50 to 1,600. Blood absorption compounds were identified by accurate mass, MS/MS ion fragment pattern, and retention time of LC and then were validated by available standard.
7
LC-MS Analysis of Acetone Dissolved Oils
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The LC-MS analysis was performed on an Agilent 1290 Infinity LC system (Thermo Finnigan, San Jose, CA, USA) coupled to Agilent 6520 Accurate-Mass Q-TOF mass spectrometer with dual ESI source. Separation of compounds was carried in an XDB-C18 column (150 mm × 2.1 mm, 3.5 µm) (Agilent Technologies, Eclipse) at a column temperature of 25 °C. Injection volume of 1.0 µL from the acetone dissolved oil samples was eluted with formic acid/water (0.1:99.9, v/v) and formic acid/acetonitrile (0.1:99.9, v/v) as the mobile phase at a flow rate of 0.5 mL/min. The total run time was 25 min including 5 min of post run. Spectra acquisition at a rate of 1.03 (spectra/s) in the Q-TOF MS detector was made in full scan (100–3200 m/z) at capillary voltage of 4000 V in positive polarity. MS parameters were set as follows: nebulizer pressure at 45 psi, skimmer 65 V, drying gas temperature at 300 °C and drying gas flow at 10 L/min. Agilent Mass Hunter Qualitative Analysis B.07.00 was used for LC-MS data analysis.
8
HPLC Analysis of Herbal Compounds
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An Agilent 1200 (Agilent Technologies, Santa Clara, CA, United States) equipped with a quaternary pump, autosampler, column oven and diode-array detector was used. Acquired data were processed using Chemstation software (Ver. B. 03. 02). Chromatographic separation was performed on an XDB C18 column (4.6 mm × 150 mm, 5 μm; Agilent, United States) at 35 °C. The mobile phase consisted of water with 0.1% trifluoroacetic acid (A) and methanol (B). The following program was used: 30% (B) for 1 min, 30%-80% (B) over 1 min-13 min and 80% (B) for 1 min, which was followed by a re-equilibration to 30% (B). The flow rate was set at 0.8 mL/min and the injection volume was 10 μL. The detection wavelengths were 240 nm for geniposide, 260 nm for berberine chloride and 275 nm for baicalin and wogonin.
9
Nanoparticle Characterization and Drug Loading
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Particle
size and PDI of NPs were measured using DLS (Zetasizer Nano ZS90,
Malvern Instruments, Malvern, UK). Morphological characteristics of
NPs were observed by SEM (JXA-840, JEOL, Tokyo, Japan). Drops of NPs
were added to the surface of the silicon wafer and dried for further
observation by SEM.
The loading capacity and EE were determined
using an HPLC (Waters Corp, Milford, MA, USA) system equipped with
an Agilent XDB-C18 column (4.6 mm × 250 mm, 5 μm) at 300
nm. Briefly, NPs were destroyed by acetonitrile on vortex for releasing
the compound, and then the mobile phase (60% of acetonitrile) was
added to extract the compound. Then compound 480 was analyzed by HPLC.
DL and EE were calculated according to the following formula
size and PDI of NPs were measured using DLS (Zetasizer Nano ZS90,
Malvern Instruments, Malvern, UK). Morphological characteristics of
NPs were observed by SEM (JXA-840, JEOL, Tokyo, Japan). Drops of NPs
were added to the surface of the silicon wafer and dried for further
observation by SEM.
The loading capacity and EE were determined
using an HPLC (Waters Corp, Milford, MA, USA) system equipped with
an Agilent XDB-C18 column (4.6 mm × 250 mm, 5 μm) at 300
nm. Briefly, NPs were destroyed by acetonitrile on vortex for releasing
the compound, and then the mobile phase (60% of acetonitrile) was
added to extract the compound. Then compound 480 was analyzed by HPLC.
DL and EE were calculated according to the following formula
10
Separating Natural Compounds via Chromatography
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Column chromatography was carried out using silica gel (3.0 × 30 cm, Qingdao Marine, China), CHP20P (3.0 × 25 cm, Mitsubishi, Japan), octadecylsilyl silica (ODS) (YMC, Japan) and Sephadex LH-20 (1.5 × 180 cm, GE Healthcare, Sweden). Semi-preparative high performance liquid chromatography (HPLC) was conducted on a 1260 system (Agilent, USA) equipped with an XDB-C18 column (9.4 × 250 mm, 5 μm, Agilent, USA). Ultra violet (UV) spectra were determined using a 241 spectrophotometer (Perkin Elmer, USA). High resolution electrosparay ionization mass spectrum (HR-ESIMS) spectra were recorded on a 6545 Q-TOF instrument (Agilent, USA). Nuclear magnetic resonance (NMR) spectra were acquired using an AV-600 instrument (Bruker, Germany). Electronic circular dichroism (ECD) spectra were obtained using a MOS-450 instrument (Bio-Logic, France). The solvents used in column chromatography were analytical grade (Tianjin Hengxing, China), the solvents used in HPLC, HR-ESIMS, ECD and activity assays were chromatographic grade (Sigma-Aldrich, USA), and the solvent used in NMR was methanol-d4 (Cambridge Isotope Laboratories, USA).
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