The LC–MS/MS system comprises an AB Sciex API5000 Tandem Mass Spectrometer, Shimadzu Prominence 20ADXR UFLC pumps and an SIL-20ACXR autosampler managed with Analyst® 1.5.1 (AB Sciex, CA, USA). The gases for the MS system were supplied by an LC–MS gas generator (Source 5000™, Parker Balston, Inc., MA, USA). The LC columns tested include Synergi polar RP (2.0 × 50 mm, 4 µm), PolymerX RP-1 (4.0 × 50 mm, 5 µm), and pen-tafluorophenyl (PFP) (2.0 × 50 mm, 2.6 µm) columns from Phenomenex, Inc., CA, USA, and Zorbax C8 (2.1 × 50 mm, 5 µm), C18 (2.1 × 30 mm, 1.8 µm) and Pursuit PFP (2.0 × 50 mm, 3 µm) columns from Agilent Technologies, Inc., CA, USA. The LC–MS/MS system was operated in a 25°C room controlled with an air conditioner. The MS conditions for PQ and the IS were optimized by separate infusion of 50 ng/ml PQ or IS into the MS at a flow rate of 10 µl/min while adjusting MS parameters to achieve maximal signal. Ionization utilized APCL+, and detection utilized multiple reaction monitoring mode. Data were processed with Analyst 1.5.1.
Zorbax c8
Manufactured by Agilent Technologies
Sourced in United States
Zorbax C8 is a reversed-phase high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of compounds. It features a C8 stationary phase bonded to silica particles, providing moderate hydrophobicity and selectivity. The Zorbax C8 column is commonly used for the analysis of pharmaceutical, environmental, and food samples.
Automatically generated - may contain errors
Lab products found in correlation
Pursuit pfp, by Agilent Technologies (1 mentions)
Synergi polar rp, by Phenomenex (1 mentions)
Polymerx rp 1, by Phenomenex (1 mentions)
Analyst 1, by AB Sciex (1 mentions)
Sciex tripletof 6600, by AB Sciex (1 mentions)
Agilent 1100, by Agilent Technologies (1 mentions)
6520 q tof mass spectrometer, by Agilent Technologies (1 mentions)
Polaris hr 3c18, by Agilent Technologies (1 mentions)
Chip cube nano esi interface, by Agilent Technologies (1 mentions)
Masshunter software, by Agilent Technologies (1 mentions)
1260 infinity 2 hplc, by Agilent Technologies (1 mentions)
Analytical balance, by Sartorius (1 mentions)
6 protocols using zorbax c8
1
LC-MS/MS Based Analytical Protocol
2
Robust Plasma Endocannabinoid Analysis
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After sampling, blood samples were immediately centrifuged, the plasma transferred into Eppendorf tubes and frozen at -60°C without any delay (Hauer et al., 2013 (link)). This procedure allows the storage of EC samples for at least 6 months (Di Marzo et al., 2009 (link)).
Plasma concentrations of the ECs anandamide (AEA), 2-arachidonoylglycerol (2-AG) as well as the N-acyl-ethanolamides (NAEs) palmitoylethanolamide (PEA), oleoylethanolamide (OEA), and stearoylethanolamide (SEA) were determined using a LC-High Resolution LC-MS/MS instrument combination (Agilent 1290/Sciex Triple TOF 6600) in positive high-resolution MRM (multiple reaction monitoring, MS resolution ∼16,000) mode. Chromatographic separation was achieved on a RP-C18 column (Zorbax C8, 2.1 mm × 50 mm, 3.5 μm; Agilent). Both components of the binary gradient contained a mixture of 2 mmol aqueous ammonium acetate and ACN at a mixing ratio of 95+5 (A) and 5+98 (B). Both buffers were moderately acidified with 0.1% acetic acid. The separation gradient started at an amount of 10% B which was linearly increased to 100% B at 10 min and was finally kept constant for another 2 min at a flow rate of 250 μl/min.
Plasma concentrations of the ECs anandamide (AEA), 2-arachidonoylglycerol (2-AG) as well as the N-acyl-ethanolamides (NAEs) palmitoylethanolamide (PEA), oleoylethanolamide (OEA), and stearoylethanolamide (SEA) were determined using a LC-High Resolution LC-MS/MS instrument combination (Agilent 1290/Sciex Triple TOF 6600) in positive high-resolution MRM (multiple reaction monitoring, MS resolution ∼16,000) mode. Chromatographic separation was achieved on a RP-C18 column (Zorbax C8, 2.1 mm × 50 mm, 3.5 μm; Agilent). Both components of the binary gradient contained a mixture of 2 mmol aqueous ammonium acetate and ACN at a mixing ratio of 95+5 (A) and 5+98 (B). Both buffers were moderately acidified with 0.1% acetic acid. The separation gradient started at an amount of 10% B which was linearly increased to 100% B at 10 min and was finally kept constant for another 2 min at a flow rate of 250 μl/min.
3
Quantifying SAM and SAH in Mouse Eyeball
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The concentration of SAM and SAH were measurement by extracting the mouse eyeball blood. According to the Zhen’s method [51 (link)], 100 μl plasma sample were extracted with 10 μl of 35% perchloric acid. Then, the acidified sample was vortexed for 1 min, subsequently, was placed at about 25 °C for 10 min, lastly, were centrifuged with the speed of 15,000 rpm at 4 °C for 10 min. Ultimately, 25 μl supernatant was analyzed using HPLC. HPLC equipment was Agilent 1100. The chromatogram column (Agilent ZORBAX C8, 250 × 4.6 mm, 5 μm) was used for the analysis. The mobile phase consisted of two solvents: solvent 1 was 8 mM octane sulphonic acid and 50 mM NaH2PO4 (PH 3.0); solvent 2 was chromatographic grade methanol. The flow rate was 0.9 mL/min. The column temperature was 35 °C. The UV detection wavelength was set at 450 nm from 0 to 8 min, and 254 nm from 8 to 16 min. The SAM/SAH standards configuring in water to different concentrations were used to trace the radio-labelled peaks.
4
Isocratic Chromatographic Separation
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The chromatogram separation was achieved by using Agilent, Zorbax C8 (250 × 4.6 mm i.d.) 5.0 μm column with a simple isocratic method. The buffer consists of 0.01 M of 1-pentane sulfonic acid and 0.02% orthophosphoric acid in purified water. Mixed buffer, acetonitrile, and methanol (800:100:100 v/v). The flow rate was 1.0 ml min−1, and injection volume was 10 μl. Detection was made at 254 nm by using a dual absorbance detector (DAD). The dissolution conditions media volume 900 ml, USP-II (Paddle), and 50 rpm were used to perform the profile.
5
LC-MS/MS Analysis of Proteins and Peptides
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Samples were analyzed by LC-MS/MS using a 6520 Q-TOF mass spectrometer controlled by Agilent MassHunter software (B.05.00 version), coupled online with a 1200 series HPLC system through a Chip Cube nano-ESI interface (Agilent Technologies, CA, USA). Chromatographic separations of intact proteins were performed on a reverse-phase, 5 μm C8 chip-column (Zorbax C8 - 0.075 mm × 40 mm, Agilent Technologies), integrating a 40 nl capacity trap-column, and a nano-spray emitter.
Peptides from tryptic digestion of GPX4 were separated on a reverse-phase, high resolution 3 μm C18 chip-column (Polaris-HR-3C18 – 0.075 mm × 150 mm, Agilent Technologies), integrating a 360 nl capacity trap-column, and a nano-spray emitter.
Peptides from tryptic digestion of GPX4 were separated on a reverse-phase, high resolution 3 μm C18 chip-column (Polaris-HR-3C18 – 0.075 mm × 150 mm, Agilent Technologies), integrating a 360 nl capacity trap-column, and a nano-spray emitter.
6
HPLC-based HCQ Dissolution Profiling
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The Agilent HPLC 1260 Infinity-II was used for the estimation of HCQ dissolution profile. It consists of four channels, pressure range up to 600 bar, degasser with an integrated purge valve, thermostatic sampler, and column compartment. The PDA detector connected to empower 3 software (Build 3471 SPs Installed: Feature Release 3 DB ID: 2639633283) to monitor the output signal. The Disteck Premiere 5100 model dissolution apparatus was used to perform the multimedia profile. The column is Agilent Zorbax C8, 250 mm × 4.6 mm i.d., 5 μm. Sartorius analytical balances were used for the weighing of standards and samples. Bio-Technics ultra sonicator is used to extract the drug from the sample matrix.
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