The samples collected in May, August, and October were dried and ground into powder. Powder samples (0.5 g) were treated for 30 min by ultrasonic extraction after soaking in petroleum ether (150 mL) for 30 min. The homogenates were filtered, and residues were immersed in 70% alcohol (20 mL) for 30 min and extracted with 70% alcohol by ultrasonic treatment for 30 min. For each extract, 2 mL was dissolved in 3.0 mL of methanol and filtered through a 0.45 µm microporous film. Then, 20 µL of filtrate was run on a Dionex-P680 HPLC (Dionex, Pliening, Germany) system with an Agilent TC-C18 column (250 mm × 4.6 mm, 5 µm, Agilent Technologies Inc., Palo Alto, AR, USA). The mobile phase consisted of acetonitrile (A solvent) and 0.1% phosphate solution (B solvent), used according to the following gradient elution program: 0 min, 0%A/90%B; 15 min, 15%A/85%B; 45 min, 15%A/85%B, 10 min, 25%A/75%B; 15 min, 25%A/75%B and 15 min, 10%A/90%B. The detection wavelength was set at 280 nm, and flow rate was 1 mL/min. The chemical standards included dihydromyricetin (15092141), myricitrin (11060222), and myricetin (14101531), which were purchased from Shanghai Yuanye Biotechnology Co., LTD, Shanghai, China, and quercetrin (20140318), which was purchased from Shanghai Jinsui Biotechnology Co., LTD.
Agilent tc c18 column
Manufactured by Agilent Technologies
Sourced in United States, Japan
The Agilent TC-C18 column is a reversed-phase liquid chromatography column designed for the separation and analysis of a wide range of organic compounds. It features a silica-based stationary phase with octadecylsilane (C18) bonding, providing high efficiency and selectivity for a variety of analytes.
Automatically generated - may contain errors
Lab products found in correlation
Pump 250b, by PerkinElmer (5 mentions)
Flexar uv vis lc spectrophotometer detector, by PerkinElmer (5 mentions)
Dihydromyricetin, by Yuanye Bio-Technology (1 mentions)
Myricitrin, by Yuanye Bio-Technology (1 mentions)
Myricetin, by Yuanye Bio-Technology (1 mentions)
Dionex p680 hplc, by Thermo Fisher Scientific (1 mentions)
Hplc system, by PerkinElmer (1 mentions)
Perkin elmer system, by PerkinElmer (1 mentions)
Agilent 1200 hplc system, by Agilent Technologies (1 mentions)
Chemstation software, by Agilent Technologies (1 mentions)
Agilent 1260 series system, by Agilent Technologies (1 mentions)
Agilent 1260 lc, by Agilent Technologies (1 mentions)
Coomassie blue staining, by Beyotime (1 mentions)
H20at, by Shimadzu (1 mentions)
Alliance 2695 model, by Waters Corporation (1 mentions)
B 540, by Büchi (1 mentions)
Agilent 1120 compact lc, by Agilent Technologies (1 mentions)
Jnm la 300, by JEOL (1 mentions)
Silica gel 60, by Merck Group (1 mentions)
Spd ultraviolet detector, by Shimadzu (1 mentions)
24 protocols using agilent tc c18 column
1
Quantification of Bioactive Compounds in Plant Samples
2
Quantitative Analysis of Paclitaxel by HPLC
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The concentration of paclitaxel was determined using HPLC fitted with a reverse phase Agilent TC C18 column (150mm ×4.6 mm, 5 μm, Agilent Technologies, CA, USA) and a UV detector capable of scanning a wavelength range of 200–600 nm. Mobile phase system comprising acetonitrile and water (60:40 v/v) at a flow rate of 1 mL/min was employed for elution. Paclitaxel was measured at a wavelength of 227 nm, and its concentration calculated using the external standard method. For this purpose, 1 mg of paclitaxel dissolved in a volumetric flask using acetonitrile to obtain a stock standard solution. The stock solution was then diluted using mobile phase to yield a series of calibration solutions analyzed by the HPLC system. A linear calibration curve was obtained over the concentration range of 0.5–25 μg/mL of paclitaxel with a regression coefficient of 0.998.
3
HPLC Quantification of Paclitaxel
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PTX quantitative determination was carried out by HPLC analysis using a pump (Perkin Elmer Pump 250B, Waltham, MA) equipped with a spectrophotometer detector (Flexar UV/Vis LC spectrophotometer detector, Perkin Elmer, Waltham, MA). A reverse phase Agilent TC C18 column (150 cm × 4.6 mm, pore size 5 μm; Agilent Technologies, Santa Clara, CA, USA) was used. The column was eluted with acetonitrile/water (60:40) at a flow rate of 1 ml/min. PTX was detected at 227 nm with a UV/vis detector. The drug concentration was calculated using the external standard method from a standard calibration curve.
4
Quantification of TMPH in Plasma and Tissues
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Plasma and tissue concentrations of TMPH were measured using a Shimadzu Liquid chromatographic 20 system equipped with a DAD(Diode Array Detector) (Shimadzu Corporation, Japan). Separations were carried out using an Agilent TC-C18 column (250 mm × 4.6 mm, 5 μm, Agilent, USA). The mobile phase consisted of water-methanol (45:55, v/v) at a flow-rate of 1 mL/min and the detector wavelength was set at 279 nm. Chromatographic separation was performed at 30 °C. The retention times of TMPH and the I.S. were 6.1 min and 10.5 min, respectively. The main validation parameters of the analytical method are in agreement with the international bioanalytical guidelines (European Medicines Agency, 2011 ; Food and Drug Administration, 2018 ) and summarized in Table 1 . For plasma analysis, the lower limit of quantification (LLOQ) for TMPH was 0.1 μg/mL and the linear range was 0.1–10 μg/mL in rat plasma. The LLOQ value of the tissue samples was 0.05 μg/mL and the method was linear over a concentration range of 0.05 to 5 μg/mL.
5
Nicotine Degradation by A. oryzae
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Resting cells of A. oryzae 112822 were prepared as previously described [35 (link)]. Nicotine degradation assay was performed by adding 5 g of resting cells to 20 mL nicotine solution (0.35 g·L− 1) and incubating at 28 °C with shaking at 150 rpm. Reaction mixtures were sampled at regular intervals and analyzed by HPLC via an Agilent Technologies 1200 series (Agilent Technologies, USA) using an Agilent TC-C18 column (150 × 4.6 mm, Agilent Technologies, USA) and an UV detector operated at a wavelength of 254 nm.
6
HPLC Quantification of Imiquimod
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For the quantitative determination of IMQ, an HPLC method was tuned. The analyses were carried out using a Perkin Elmer system (Perkin-Elmer, Shelton, CT, USA) under isocratic conditions. Analyses were performed using an Agilent TC C18 column (250 mm × 4.6 mm × 5 µm; Agilent Technologies, Santa Clara, CA, USA) tempered at room temperature. The mobile phase consisted of acetonitrile:acetate buffer (pH 4.0, 0.05 M):triethylamine (30:69.85:0.15 v/v), the flow rate was 1 mL/min and the detection wavelength was set a 242 nm. Before use, the mobile phase was filtered through a 0.45-μm-pore-size membrane filter and degassed.
The external standard method was used for the calculation of the drug content. For this purpose, about 1 mg of IMQ was weighted and dissolved in methanol in a volumetric flask to get a stock solution. This solution was diluted in the mobile phase, providing a series of calibration solutions, subsequently injected into the HPLC system (Perkin-Elmer, Shelton, CT, USA). The calibration curve was created by plotting the IMQ standard peak area vs. the corresponding drug concentration. A linear calibration curve was obtained in the 0.5–25 μg/mL concentration range with a regression coefficient of 0.999.
The external standard method was used for the calculation of the drug content. For this purpose, about 1 mg of IMQ was weighted and dissolved in methanol in a volumetric flask to get a stock solution. This solution was diluted in the mobile phase, providing a series of calibration solutions, subsequently injected into the HPLC system (Perkin-Elmer, Shelton, CT, USA). The calibration curve was created by plotting the IMQ standard peak area vs. the corresponding drug concentration. A linear calibration curve was obtained in the 0.5–25 μg/mL concentration range with a regression coefficient of 0.999.
7
In vitro Enzymatic Assay of MilA
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In vitro assays of recombinant MilA were carried out at 37 °C for 1 h in a total volume of 100 μl that contained Tris–HCl buffer (100 mM, pH 7.5), paraformaldehyde (15 mM), 2-mercaptoethanol (50 mM), tetrahydrofolate (2 mM, pH 7.5), CMP and dCMP (1 mM, pH 7.5) and the corresponding His-tagged MilA or its mutants (10 μg). The reactions were quenched by the addition of trichloroacetic acid (4%) on ice, the products were resolved by Agilent TC-C18 column (4.6 mm × 250 mm, 5-Micron) on an Agilent 1200 HPLC system using a mobile phase of a gradient of methanol in water supplied with formic acid (0.1%). The constant flow rate for the LC eluent is 0.3 ml/min. Chromatograms were detected using the absorbance at 275 nm. The percentages of methanol (M) at time t varied according to the following scheme: (t, M), (0, 3), (30, 3), (31, 90), (35, 90), (36, 3), (45, 3). The accurate mass of the reaction products that were previously determined by NMR12 (link) were analyzed by QTOF/MS (Agilent G6530A).
8
Quantification of MEB55 and ST362 using HPLC
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The concentrations of MEB55 and ST362 were quantified using an HPLC system consisting of a PerkinElmer PUMP 250B, equipped with a Flexar UV/Vis LC spectrophotometer detector (PerkinElmer, Waltham, MA, USA). We utilized a reversed phase Agilent TC C18 column (150 mm × 4.6 mm, pore size 5 μm; Agilent Technologies, Santa Clara, CA, USA). The mobile phase consisted of a mixture of acetonitrile and water (85:15 v/v), which was degassed and pumped through the column at a flow rate of 1 mL/min. The ultraviolet detector was set at 294 nm and 300 nm for MEB55 and ST362, respectively. The SL concentrations were calculated using a calibration curve and an external standard. A total of 1 mg of MBE55 or ST362 was placed in a volumetric flask and dissolved in acetonitrile to obtain a standard stock solution. This solution was then diluted in the mobile phase to generate the series of standard solutions. Linear calibration curves were obtained over a concentration range of 0.5−25 μg/mL. Both compounds had regression coefficients of 0.999.
9
HPLC Analysis of Samples
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HPLC analyses were performed on an Agilent 1260 series system (Agilent Technologies, USA) consisting of a quaternary pump, online vacuum degasser, autosampler, thermostated column compartment and DAD full-wavelength scanning detector. An Agilent TC-C18 column (150 mm × 4.6 mm i.d., 5.0 μm particle size) from Agilent Technologies (USA) was used for all chromatographic separations. A linear gradient elution of Eluents A (0.1% (v/v) aqueous phosphoric acid) and B (0.1% (v/v) phosphoric acid in acetonitrile) was used for the separation. The elution program was well optimized and conducted as follows: the first linear gradient was 5% Eluent B in the range of 0 ~ 3 min, the second one was 5% ~ 12% Eluent B in the range of 3 ~ 8 min, the third one was 12% ~ 15% Eluent B in the range of 8 ~ 11 min, the fourth one was 15% Eluent B in the range of 11 ~ 25 min and the last one was 15% ~ 100% Eluent B in the range of 25 ~ 35 min. Then, the system was restored to the initial conditions after 5 min. The solvent flow rate was 1.0 mL/min, the detection wavelength was set at 210 nm, the column temperature was maintained at 25 °C and the injection volume was 20 μL. Chemstation software (Agilent Technology) was used for peak detection and peak area calculation.
10
HPLC Analysis of TBF Fractions
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The components of the TBF fraction were determined as described elsewhere [23 (link)]. The TBFs were analyzed using a high-performance liquid chromatography (HPLC) system (Agilent 1260 LC, Agilent Technology Co., Ltd.) equipped with an Agilent TC-C18 column (4.6 mm × 250 mm, Agilent Technology Co., Ltd.). The sample was eluted using mobile phase A of water and mobile phase B of a water solution containing 95% (v/v) methanol. A linear gradient program was applied as follows: 20% (v/v) B for 0–5 min, 20–40% (v/v) B for 5–15 min, and 40% (v/v) B for 15–40 min. The detection wavelength was set at 260 nm. Pure rutin and quercetin were used as standards for HPLC analysis.
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