Supernatants from monocytes stimulated for 24 hours with 0.5 MOI PA were processed to measure concentrations of HODEs and hydroxyeicosatetraenoic acids (HETEs) using method adopted from Imaizumi et al.14 (link) LC/MS/MS performed using 4000 QTRAP (Applied Biosystems, Foster City, CA, USA) with electrospray ionization (ESI) source. High-performance liquid chromatography (HPLC) system used Agilent 1200 series LC pump with thermostatted autosampler (Agilent Technologies, Santa Clara, CA, USA). Chromatography performed using Luna C-18(2) column (3 µm particle, 150×3.0 mm). Data acquisitions and instrument control done with Analyst 1.4.2 software (Applied Biosystems). Detection accomplished with multiple reaction monitoring modes with negative ion detection. Collision energy, declustering potential, and collision cell exit potential optimized for each compound to obtain optimum sensitivity. Transitions monitored were mass-to-charge ratio (m/z): m/z 295.1→194.8 for 13-HODE; 295.0→171.0 for 9-HODE; 319.1→219.0 for 15-HETE; 319.1→115.0 for 5-HETE; 319→179 for 12-HETE.
Thermostatted autosampler
Manufactured by Agilent Technologies
Sourced in United States
The Thermostatted autosampler is a lab equipment product that provides temperature-controlled sample handling. It automatically introduces samples into an analytical instrument for analysis, maintaining consistent sample temperature to ensure reliable and reproducible results.
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Lab products found in correlation
3 protocols using thermostatted autosampler
1
Quantification of Lipid Mediators
2
Quantification of Citric Acid in Lime Juice
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Lime juice samples were centrifuged at 5000× g for 10 min at 4 °C, filtered using syringe filters (0.2 µm, VWR International, PR, USA) and diluted ten times with Milli-Q water for analysis. A HPLC method was developed using a 1200 series HPLC system equipped with a quaternary pump and a thermostatted autosampler (Agilent Technologies, Santa Clara, CA, USA). Citric acid was quantified in triplicate using a diode array detector (Agilent 1100/1200) monitored at 210 nm and an Aminex HPX-87H ion exclusion column at a controlled temperature of 55 °C. The analytical conditions were as follows: mobile phase 0.045 N H2SO4/acetonitrile (94:6; v/v), flow 0.3 mL/min, and detector temperature 35 °C [23 (link)].
3
Quantitative Chromatographic Analysis of Compounds
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Quantitative chromatographic analyses were performed using an Agilent 1260 liquid chromatography system equipped with a vacuum degasser, a quaternary pump, a thermostatted autosampler, a column temperature controller and a diode array detector (Agilent Technologies, Palo Alto, CA, USA) at 45 °C on a Kinetex ® 2.6 µm C18 100 Å LC column (30 × 3 mm) with a mobile phase flow rate of 1.1 mL / min. Composition of mobile phase A was 0.1 % (v/v) formic acid in water, B was MeCN/water 95/5 (v/v) with 0.1 % (v/v) of formic acid. A 3.91 min long, linear gradient program was applied: 0 % B in the first 0.3 min, 0-100 % B between 0.3 and 1.8 min, then 100 % B was kept for another 0.6 min, and finally at 2.41 min the percentage of B was dropped to 0 %. This was followed by an equilibration period of 1.5 min prior to the next injection. Chromatograms were recorded at the wavelength of 200-500 nm, integration was carried out at the UV max of each compound. The applied injection volume was 6 μL. ChemStation B.04.03 was used for data acquisition and analysis.
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