Paclitaxel was quantified by HPLC with UV detection (Shimadzu Nexera-i lc-2040c 3D, Shimadzu Corporation, Kyoto, Japan). Column: C18, 5 μm, 25 cm x 4.6 mm. Mobile phase: 45% phosphate buffer 0.005 M and 55% acetonitrile, 1 ml/min. Detection: 230 nm. Column temperature: 35°C. At least 20 μl of the supernatant was injected into the HPLC unit. A standard curve (paclitaxel) was established during the same run (concentration between 5 and 500 μg/ml).
Nexera i lc 2040c 3d
Manufactured by Shimadzu
Sourced in Japan
The Nexera-i LC-2040C 3D is a high-performance liquid chromatography (HPLC) system from Shimadzu. It is designed to provide efficient and reliable liquid chromatography analysis. The Nexera-i LC-2040C 3D features a compact and modular design, allowing for easy customization and integration into various laboratory workflows.
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10 protocols using nexera i lc 2040c 3d
1
Quantitative HPLC Analysis of Paclitaxel
2
Evaluating CYP3A Activity in iPS-hep
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CYP3A activity in iPS-hep on day 30 of differentiation was evaluated. PHH plated for 4 h or 48 h were used as references. iPS-hep or PHH cultures were washed with warm William’s E media supplemented with PS, L-Gln, and Primary Hepatocyte Maintenance Supplements (CM4000, ThermoFisher). Assays were started by adding media containing 5 μM midazolam (Wako). The volume of media added was 500 μL to the PHH, 300 μL to the upper, and 600 μL to the lower chamber. 0.5, 1, and 2 h, 60 μL of supernatants were collected from the upper and lower chamber and kept at −80℃ until LC–MS/MS analysis of the metabolite, 1’-OH midazolam. For iPS-hep, midazolam was added to both the upper and lower chambers. Supernatants from both the upper and lower chambers were collected and combined. The collected supernatants were extracted by adding acetonitrile containing an internal standard, 1 ‘-OH midazolam 13C3. LC–MS/MS quantified the metabolites with the liquid chromatography Nexera I LC-2040C 3D (Shimadzu) and the mass spectrometer LCMS-8050 (Shimadzu), using Inertsil ODS-3, 2.1 × 33 mm, 3 µm column (GL Sciences) (Supplemental Table S2). The protein amount per well was quantified using the Bradford protein assay. Metabolite concentrations were normalized with protein amounts.
3
HPLC Analysis of Penetration Enhancers
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The HPLC system comprised a Shimadzu Nexera-i LC-2040C 3D equipped with a photodiode-array detector. Post-run analysis was performed using Labsolutions 5.82 (Shimadzu). All HPLC methods have been validated and previously described [13] (link). As the penetration enhancers used in this study could potentially interfere with the analysis, specificity was investigated. Blank samples containing 100% penetration enhancer, samples of individual penetration enhancers spiked with known quantities of each of the chemicals, and samples run in unspiked ARS were injected onto the column to ensure no interference with the chemical peak occurred. All methods remained specific for the determination of the respective chemicals and no interference was detected, thus specificity was assured. HPLC methods were therefore used as previously reported. Calibrations were performed daily (r2 > 0.999), and all samples measured in triplicate.
4
Quantifying Costunolide Loading in MSNs
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The drug loading of MSNs-COS and MSNs-COS-MAC was determined by HPLC (Nexera-i LC-2040C 3D, Shimadzu, Japan). The chromatographic conditions used in the analysis were as follows: a Shim-pack GIST C18 column (50 mm × 2.1 mm, 2 μm, Shimadzu), a mobile phase of a methanol/pH 2.0 phosphoric acid solution (70:30), a flow rate of 0.3 mL/min, a detection wavelength of 225 nm, a column temperature of 25 °C and an injection volume of 5 μL. COS in the drug-loaded samples was extracted with methanol under an ultrasonic condition and then filtered using a 0.22 μm membrane filter before running the HPLC analysis. Drug loading (%) = (Weight of costunolide in samples /Weight of samples) × 100.
5
Quantification of Bioactive Compounds in P. coronarius Extract
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Identification and quantification of the bioactive compounds from P. coronarius extract were performed by using a Shimadzu Nexera-i LC–2040C 3D plus liquid-chromatograph system equipped with a photodiode array detector (PDA). A Phenomenex C18 (2) 100 A, 150 mm × 4.6 mm × 5 µm column was selected, and it was kept at 30 °C temperature. The mobile phases used for elution contained methanol (A) and formic acid 0.1% (B). The gradient program used was: 5% A and 95% B from 0 to 3 min, 25% A and 75% B from 3 to 6 min, 37% A and 63% B from 9 to 13 min, 54% A and 46% B from 18 to 22 min, 95% A and 5% B from 26 to 29 min and 5% A and 95% B from 30 to 36 min. The flow rate was 0.5 mL/min, and the injection volume was 10 µL. The detection was performed at multiple wavelengths: 254, 270, 275, 326, 337 and 360 nm. The polyphenols from the extract were identified by comparing the retention times from the extract chromatograms with the ones from the standard-solution chromatograms.
The bioactive-compound content was determined with the following equation:
The bioactive-compound content was determined with the following equation:
6
Quantifying Paclitaxel in Balloon Catheters
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Unused and used DCBs were placed in cryovials. Balloons were extracted and paclitaxel content was quantified by high-pressure liquid chromatography (HPLC) with UV detection (Shimadzu Nexera-i LC-2040C 3D, Shimadzu Corporation, Kyoto, Japan) as described before [14 (link)].
7
HPLC Analysis of IMB16-4 Drug Loading
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The samples were analyzed by HPLC (Nexera-i LC-2040C 3D, Shimadzu, Japan). Analysis was carried out on a Shim-pack GIST C18 column (50 × 2.1 mm, 2 μm, Shimadzu). The mobile phase consisted of an 80:20 (% v/v) mixture of methanol and pH 2.0 phosphoric acid solutions, the flow rate was 0.3 mL/min and the detection wavelength was 258 nm. The column temperature was 25 °C. The injection volume was 5 μL. The retention times are about 3.07 min for IMB16-4. The samples were filtered using a 0.22 μm membrane filter before running the HPLC analysis. Drug loading (%) = (weight of IMB16-4 in samples/weight of samples) × 100.
8
Quantification of Plasmid DNA using HPLC
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The plasmid DNA was extracted using alkaline lysis and quantified using the analytical high-performance liquid chromatography (HPLC) method based on hydrophobic interaction chromatography (HIC), as fully described by Soares et al. [7 (link)]. A 4.6/100 mm HIC source of 15 PHE PE columns (Amersham Bioscience, Uppsala, Sweden) that was connected to an HPLC system (Shimadzu, Nexera-i LC-2040C 3D, Duisburg, Germany) was used. The plasmid concentration was calculated using calibration curves that were constructed from the serial dilutions of the plasmid DNA standards (ranging from 0.04 to 20 µg mL−1). Then, the corresponding plasmid copy number (PCN) was calculated using Equation (3) [33 (link)]:
where the size of plasmid pFM23 was 6053 bp.
where the size of plasmid pFM23 was 6053 bp.
9
Quantification of Paclitaxel in Balloon Catheters
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For analysis of paclitaxel each balloon was placed in a vial and cut off from the catheter shaft, and a defined volume of acetonitrile was added. The vials were firmly closed and intensely shaken on a vortexer for at least 30 seconds followed by treatment in a ultrasound bath for 30 minutes and centrifugation for 10 minutes (16,000 x g Eppendorf microcentrifuge 5415C). Samples with a high concentration of analyte were diluted with acetonitrile.
Paclitaxel was quantified by HPLC with UV detection (Shimadzu Nexera-i Lc-2040c, 3D, Shimadzu Corporation, Kyoto, Japan). Column: C18, 5 μm, 25 cm x 4.6 mm. Mobile phase: 45% phosphate buffer 0.005 M and 55% acetonitrile, 1 ml/min. Detection: 230 nm. Column temperature: 35°C. At least 20 μl of the supernatant was injected into the HPLC unit. A standard solution (paclitaxel) was injected during the same run (concentration approximately 50 μg/ml ± 5%).
Paclitaxel was quantified by HPLC with UV detection (Shimadzu Nexera-i Lc-2040c, 3D, Shimadzu Corporation, Kyoto, Japan). Column: C18, 5 μm, 25 cm x 4.6 mm. Mobile phase: 45% phosphate buffer 0.005 M and 55% acetonitrile, 1 ml/min. Detection: 230 nm. Column temperature: 35°C. At least 20 μl of the supernatant was injected into the HPLC unit. A standard solution (paclitaxel) was injected during the same run (concentration approximately 50 μg/ml ± 5%).
10
Validated HPLC Analytical Methods
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The HPLC system was a Shimadzu Nexera-i LC-2040C 3D with photodiode-array detector. Postrun analysis was performed using LabSolutions 5.82 (Shimadzu). All HPLC methods used have been previously described and validated (Llewelyn et al. 2018; summarized in Table 2 ). All samples were run in triplicate.
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