RP-HPLC was performed using a Varian Pursuit C18 column (50 mm × 2.1 mm (ID)), 300 Å pore size, Varian, Palo Alto, CA). For C-peptide isolation, a linear gradient of acetonitrile was employed, the details are given in Section 3.3.
Pursuit c18 column
Manufactured by Agilent Technologies
Sourced in United States
The Pursuit C18 column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of compounds. It features a stationary phase of octadecylsilane (C18) bonded to silica gel, which provides efficient analyte retention and separation. The column is suitable for use in various HPLC applications, including pharmaceutical, environmental, and food analysis.
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Lab products found in correlation
Agilent hplc system, by Agilent Technologies (2 mentions)
Api 5000, by AB Sciex (1 mentions)
Agilent 1260 series, by Agilent Technologies (1 mentions)
Waters 2487 dual absorbance diode array detector, by Waters Corporation (1 mentions)
Chromafil xtra ptfe 45 13, by Macherey-Nagel (1 mentions)
Magna lyser, by Roche (1 mentions)
Agilent chemstation software, by Agilent Technologies (1 mentions)
7 protocols using pursuit c18 column
1
RP-HPLC for C-peptide Isolation
2
Melittin Purification from Recombinant Fusion
3
Brensocatib Plasma Concentration Analysis
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The plasma concentrations of brensocatib were analyzed using a validated LC‐MS/MS with dipotassium ethylenediaminetetraacetic acid as an anticoagulant. The chromatographic method consisted of a Pursuit C18 column (Agilent Technologies, Santa Clara, California) and a gradient mobile phase of 0.1% formic acid in acetonitrile/water, with 13C6‐brensocatib as an internal standard. Brensocatib and the internal standard were monitored by the precursor and product ions of m/z 421.2→100.1 for brensocatib and m/z 427.2→100.1 for the internal standard using an API 5000 or API 5500 LC‐MS/MS (Sciex, Framingham, Massachusetts). The assay range was 0.250 to 150 ng/mL, with overall precision and accuracy for intra‐ and interassays of 2.2% to 5.9% and −2.7% to 0.3%, respectively.
4
Mass Spectrometry Peptide Characterization
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Purified peptides were characterized by high-resolution mass spectrometry using a Thermo LTQ-FTMS instrument (Thermo Fisher Scientific, Waltham, MA, USA) or an Agilent 6545XT LC-QTOF (Agilent Technologies Inc., Santa Clara, CA, USA). The mass-to-charge ratios were used to determine the experimental mass of the peptide, which was verified against the calculated mass. Analytical RP-HPLC spectra were obtained using an Agilent 1260 series with a Pursuit C18 column (4.6 mm × 250 mm, 5 μm). Purified peptides were prepared in 5% MeCN in H2O (0.1% TFA), and then run on a 5–95% MeCN gradient (0.1% TFA) over 10 min at 1 mL/min. Peptide purity was determined to be >95% via integration at 220 nm.
5
HPLC Analysis of Endogenous Salicylic Acid
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The total endogenous SA was analyzed by high-performance liquid chromatography (HPLC) [66 (link)]. The seedlings (0.2–0.3 g) were extracted using 20 mL of dH2O (90–100 °C) and incubated at 100 °C for 30 min with subsequent cooling. Membrane filters (0.45 µm) (Chromafil Xtra PTFE–45/13, Macherey-Nagel GmbH Co, Duren, Germany) were used to filter the extracts. The analysis was caried out using a Waters Breeze chromatograph (Waters Corporation, Milford, MA, USA) with a Waters 2487 Dual & Absorbance diode array detector at 305 nm. A Pursuit C18 column (250 × 4.6 mm, 5 µm) (Agilent Technologies, Santa Clara, CA, USA) was used. As a mobile phase, 0.5% H3PO4: acetonitrile = 65:35 (1.0 mL min−1) was used. A total of 20 µL of the extract was injected into the chromatography system using a Waters 2707 automated sampler (Waters Corporation, Milford, MA, USA). The software calibration curve was used to calculate the total SA content.
6
Quantification of GSH and GSSG in Tissues
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After isolation, heart left ventricle and liver tissues were weighed and put directly into homogenization bead tubes containing 125 mM sucrose, 5 mM TRIS, 1.5 mM EDTA, 0.5%TFA and 0.5%MPA in mobile phase. A MagNA lyser (Roche, USA) was used to homogenize the tissue. Then samples were spun at 14000xg at 4 °C for 20 min. Supernatants were collected and either analyzed directly using an Agilent HPLC system equipped with a Pursuit C18 column (150 ×4.6 mm, 5 µm; Agilent Technologies) operating at a flow rate of 1 ml/min or stored at −80 °C for later analysis. The mobile phase consisted of 0.09% trifluoroacetic acid diluted in ddH2O and mixed with HPLC-grade methanol in a 90:10 ratio. Standard solutions were used to estimate the retention times for GSH and GSSG. Using Agilent Chemstation software, absolute amounts of GSH and GSSG were acquired by integrating the area under the corresponding peaks, and values were calculated from standard curves.
7
Quantitative HPLC Analysis of GSH and GSSG
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Human and mouse brain specimens as well as pelleted CHO cells were homogenized in buffer containing 125 mM sucrose, 5 mM TRIS, 1.5 mM EDTA, 0.5% trifluoroacetic acid (TFA) and 0.5% mycophenolic acid (MPA) in mobile phase. Samples were spun at 14,000×g at 4 °C for 20 min. Supernatants were collected and analyzed using an Agilent HPLC system equipped with a Pursuit C18 column (150 × 4.6 mm, 5 µm; Agilent Technologies) operating at a flow rate of 1 mL/min. The mobile phase consisted of 0.09% TFA diluted in ddH2O and mixed with HPLC-grade methanol in a 90:10 ratio. Standard solutions were used to estimate the retention times for GSH and GSSG. Using Agilent Chemstation software, the absolute amounts of GSH and GSSG were calculated by integrating the area under the corresponding peaks, and values were calculated from standard curves.
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