The all-trans isomer of (3S,3′S)-AST was prepared from the crude extracts of the total flower on an Agilent 1200 HPLC system (Agilent Technologies Inc., Santa Clara, CA, USA) with an InertSustain C18 column (5 μm, 250 mm × 10 mm, GL Science, Tokyo, Japan) [27 (link)]. The oily flowers extract is provided by the manufacturer settled in Weifang, China. The binary mobile phase consisted of A: 0.1% trifluoroacetic acid in water (v/v) and B: 95% methanol mixed with 5% acetonitrile (v/v). The solvent gradient was as follows: 0–5.8 min, 60–80% B; 5.8–32.6 min, 80–100% B; 32.6–35 min, 100% B; 35–47 min, 100–60% B; 47–50 min, 60% B. Peaks were detected at 470 nm. The flow rate was set at 3.0 mL/min. Under these conditions, the fraction containing the all-trans isomer of (3S,3′S)-AST eluted at a retention time of 34 to 35 min and was collected and dried under a nitrogen stream.
Inertsustain c18 column
Manufactured by GL Sciences
Sourced in Japan, United States
InertSustain 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 chemically bonded C18 stationary phase and is engineered to provide reliable and consistent performance.
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Lab products found in correlation
Ultimate 3000sd, by Thermo Fisher Scientific (2 mentions)
Sil 30ac autosampler, by Shimadzu (2 mentions)
Q exactive hybrid quadrupole orbitrap mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Dionex ultimate 3000 lc system, by Thermo Fisher Scientific (2 mentions)
Lcms 8040 uhplc system, by Shimadzu (2 mentions)
Lc 20a, by Shimadzu (2 mentions)
Hplc system, by Shimadzu (2 mentions)
Agilent 1200, by Agilent Technologies (1 mentions)
High performance liquid chromatography (hplc), by Shimadzu (1 mentions)
1200 series, by Agilent Technologies (1 mentions)
Dimethyl sulfoxide (dmso), by Bio-Techne (1 mentions)
Radioimmunoprecipitation buffer, by Nacalai Tesque (1 mentions)
Triple quad 5500 system, by AB Sciex (1 mentions)
Lc 20ad system, by Shimadzu (1 mentions)
Cut off centrifugal filter unit, by Merck Group (1 mentions)
Lc system, by Shimadzu (1 mentions)
Lcms 8040 triple quadrupole mass spectrometer, by Shimadzu (1 mentions)
Labsolution, by Shimadzu (1 mentions)
Lcms 8060, by Shimadzu (1 mentions)
2010 hplc, by Shimadzu (1 mentions)
47 protocols using inertsustain c18 column
1
Isolation of all-trans (3S,3'S)-AST
2
EGFR TKI Uptake in ABCG2 Expressing Cells
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Flp-In-293/ABCG2 WT cells, Flp-In-293/ABCG2 Q141K cells, and Flp-In-293/mock cells were harvested by trypsinization and suspended in DMEM at 1.0 × 106 cells/mL. Then, an EGFR TKI was added to the suspension at 20 µM, and the cells were incubated in a water bath at 37 °C or 4 °C for 0, 1, 5, 10, 30, or 60 min. After incubation, the cells were washed twice with cold PBS, 0.01 N NaOH was added, and sonication was performed to prepare lysates.
The concentration of each EGFR TKI in the lysates was measured by HPLC using an LC-2000Plus system with a PU-2089 Plus pump, CO-2067Plus column oven, UV-2075Plus UV detector, and InertSustain C18 column (5 μm 4.6Φ × 150 mm; GL Sciences, Inc., Tokyo, Japan). The mobile phase was a mixture of 0.1 M TEA-H3PO4 (pH 8): AcCN: THF at 55:45:2 (v/v/v) for gefitinib, erlotinib, and afatinib, while a 45:55:2 (v/v/v) mixture was used for lapatinib. The flow rate was 1.0 mL/min and the temperature was 30 °C. Detection of gefitinib, erlotinib, and afatinib was performed at 338 nm, while lapatinib was detected at 260 nm.
The concentration of each EGFR TKI in the lysates was measured by HPLC using an LC-2000Plus system with a PU-2089 Plus pump, CO-2067Plus column oven, UV-2075Plus UV detector, and InertSustain C18 column (5 μm 4.6Φ × 150 mm; GL Sciences, Inc., Tokyo, Japan). The mobile phase was a mixture of 0.1 M TEA-H3PO4 (pH 8): AcCN: THF at 55:45:2 (v/v/v) for gefitinib, erlotinib, and afatinib, while a 45:55:2 (v/v/v) mixture was used for lapatinib. The flow rate was 1.0 mL/min and the temperature was 30 °C. Detection of gefitinib, erlotinib, and afatinib was performed at 338 nm, while lapatinib was detected at 260 nm.
3
Profiling Phenolic Compounds in Plants
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Phenolic acids and flavonoids were extracted as described previously by Chumroenphat, Somboonwatthanakul, Saensouk, and Siriamornpun (2021) (link) and analyzed by HPLC (Shimadzu, Kyoto, Japan) following the protocol of Kubola, Siriamornpun, and Meeso (2011) (link). Briefly, samples (1.0 g) were extracted at 37 °C for 12 h with 20 mL of HCl/methanol (1:100, v/v) solution while being shaken at 150 rpm in the dark. Following filtering, the pellet performed a second extraction, and the mixed filtrates were dried under vacuum at 40 °C. Prior to HPLC analysis, the residue was redissolved in 5 mL of methanol/water (1:1, v/v) and filtered through a 0.45 µm nylon membrane. The phenolics were separated by an InertSustain® C18 column (250 mm × 4.6 mm, 5 µm; GL Sciences Inc., Tokyo, Japan). The wavelengths for detecting hydroxybenzoic acids, hydroxycinnamic acids, and flavonoids were 280, 320, and 370 nm, respectively. The phenolics in the extracted samples were identified according to the retention time and spectrum of each standard.
4
HPLC Analysis of Burdock Root Constituents
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High performance liquid chromatography (HPLC) was used to screen the chemical constituents in BD root aqueous extract. The HPLC was performed using an Agilent Technologies 1200 series (Agilent, Santa Clara, CA, USA). 10 mg of aqueous extract of burdock root powder was dissolved in 1 ml deionized water and sample was introduced into InertSustain C18 column (250 × 4.6 mm, 5 μm) (GL Sciences, Nakano-ku, Tokyo, Japan). Conditions included a flow rate 1.0 mL/min in elution mode gradient using mixture of acetonitrile and 0.1% acetic acid. Gradient conditions included acetonitrile (25%) and 0.1% acetic acid (75%) at initial 10 min, then acetonitrile (50%) and 0.1% acetic acid (50%) for the next 10 mine, and acetonitrile (25%) and 0.1% acetic acid (75%) for the last 10 mine. The injection volume of the aqueous sample was 10 μL and a run time of 30 min. Chlorogenic acid (1 mg/ml in DMSO, Tocris, Bristol, UK) was used for identification.
5
Quantifying Thyroid Hormones in Mice
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Organ-specific T4, T3, and TRIAC contents were measured using LC-MS/MS.18 (link) We mechanically homogenized 50 mg of either the liver or cerebrum of a mouse in 300 μL radioimmunoprecipitation buffer (Nacalai Tesque) and left the homogenate on ice for 30 min. Supernatants were centrifuged at 10,000 g for 10 min at 4°C and collected in microtubes. We added 300 μL methanol and 600 μL chloroform, mixed with vortex mixer, and centrifuged at 15,000 g for 2 min at 4°C. The upper water/methanol phase was collected in new microtubes. We injected 6 μL of each sample into a Triple Quad 5500+ system and QTRAP Ready (AB SCIEX). Chromatography was performed using InertSustain C18 column (2.1 × 150 mm, 5 μm; GL Sciences, Tokyo, Japan) maintained at 40°C. A gradient of mobile phase A (0.5 mM ammonium fluoride in water) and mobile phase B (methanol) was used. The general conditions were as follows: 40% methanol (0–1 min), 40–90% methanol linear gradient (1–10 min), 90% linear gradient (10–15 min); flow rate of 0.2 mL/min. The following MS settings were adopted: curtain and collision gas pressure of 40 and 8 psi, respectively; ion spray voltage of −4500 V; temperature of 500°C; and ion source gas 1 and 2 pressure of 80 and 70 psi; correspondingly. T4, T3, and TRIAC were all measured with ESI in negative mode by MRM. The limits of quantification of T4, T3, and TRIAC were all 0.01 ng/mL.
6
Comprehensive Comparative HPLC Analysis of Camellia Tea
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For comparison upon components and contents of tea extracts obtained via different extraction solvents, water extract and methanol extract of Camellia GT at the same mass concentration were analyzed by HPLC. The HPLC system was accomplished with a Chromaster-5110 single pump, Chromaster-5260 auto sampler, and Chromaster-5420 UV–VIS Detector (Hitachi High-Tech Sci. Corp., Japan) using an InertSustain C18 column (5 μm, 4.6 × 250 mm, GL Sciences Inc., Japan) under a mode of gradient eluent. The gradient used was mobile phases A (3% acetic acid solution, HAc: water =3:97) and mobile phases B (methanol), where the percentage of mobile phases A was changed over time as follows: 0–1 min, 100%; 1–28 min, 100–37%; 28–33 min, 37–100%. The rejection volume was 10 μL with the flow rate of 1 mL min−1. The chromatographic separation was completed in 33 min for each sample. The separated components were detected at 280 nm using the Chromaster-5420 UV–VIS Detector. In fact, as Cabrera et al. [36] (link) recommended, this is more reliable, rapid and simpler method of HPLC for simultaneous determination of catechins, gallic acids (GA) and caffeine (CAF).
7
HPLC Analysis of Chemical Components in BF
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HPLC is a common and effective analytical method for component identification. In this study, we identified the chemical components of BF by using HPLC. An LC-20AD system (Shimadzu Corp, Kyoto, Japan) equipped with an InertSustain C18 column (4.6 mm × 250 mm, 5 μm, GL Science Inc., Tokyo, Japan) at 30°C was used for analysis. The mobile phase was composed of A (methanol) and B (0.1% formic acid in water). The following gradient elution was used: 0–10 min, 5% A; 10–15 min, 10%–16% A; 15–20 min, 16%–30% A; 20–25 min, 30%–32% A; 25–40 min, 32% A; 40–45 min, 32%–72% A; 45–60 min, 72%–75% A; 60–65 min, 75%–5% A. UV absorption was determined at 280 nm, the injection volume was 10 μL, and the flow rate was 1.0 ml/min.
8
Enzymatic Activity of SPY Variants
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The in vitro enzyme activity of wild type and mutant SPY were analyzed using a sensitive HPLC method described previously68 (link) with slight modification. Specifically, the reaction mixture consisted of 5 μg tag-removed enzyme, 60 μM GDP-fucose, 50 mM Tris pH 8.0, 300 mM NaCl, and 5 mM MgCl2 in a total volume of 40 μl. The reactions were undertaken at room temperature for 1 h and then quenched at 95 °C for 90 s. HPLC analyses were performed with a InertSustain C18 column (5 μm particle size, 250 mm × 4.6 mm, GL science) on an EClassical 3100 series apparatus, equipped with a high-pressure constant current pump (P3100), a well-plate autosampler (S3100), a thermostatted column compartment (O3100) and a variable wavelength detector (DAD3100). The mobile phase consisted of phosphate buffer (10 mM phosphate buffer, pH 3.95) running at a flow rate of 1 mL/min, and the detector was set at 254 nm. The standard GDP and GDP-fucose were used as references.
9
Human TDO Purification and Analysis
10
Lyso-Gb3 Quantification in Plasma
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Lyso-Gb3 in plasma was measured by means of liquid chromatography-tandem mass spectrometry (LC-MS/MS) according to the method described previously [24 (link)]. Various concentrations of Lyso-Gb3, ranging from 0.08 to 250 nmol/L, and stable-isotope labeled Lyso-Gb3 [25 ], which has one 13C and three deuteriums, were used as the standard and internal standard, respectively. For the LC, a LC system (Shimadzu, Kyoto, Japan) and an InertSustain C18 column (30 mm × 3.0 mm I.D., 5 μm: GL Science, Tokyo, Japan) were used. For detection of Lyso-Gb3, a LCMS-8040 triple quadrupole mass spectrometer (Shimadzu, Kyoto, Japan) equipped with an electrospray ionization interface was used in the positive-ion mode. The Multiple Reaction Monitoring (MRM) conditions were optimized with an automatic MRM optimization function. In the MRM mode, the following transitions were monitored: m/z 786.8 → 282.3 for Lyso-Gb3 and m/z 790.8 → 286.2 for the internal standard.
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