Bond elut certify 2 column

The preparation and handling of samples, reference standards, and HPLC–MS measurements were performed as described elsewhere [32 (link),33 (link)]. Plasma samples (200 µL), added with 300 µL of 10M sodium hydroxide (NaOH), were subjected to alkaline hydrolysis at 60 °C for 30 min. The sample pH was then adjusted to six using 300 µL 58% acetic acid. The prepared samples were then subjected to solid-phase extraction (SPE) using a Varian Bond Elut Certify II column. The extracted metabolites were evaluated by LC–MS/MS using an Agilent 6460 Triple Quad mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) and an Agilent 1200 high-performance liquid chromatography (HPLC) system (degasser, binary pump, well-plate sampler, thermostatic column chamber). A Phenomenex Kinetex column (150 mm 2.1 mm, 2.6 m; Phenomenex, Aschaffenburg, Germany) was used in the HPLC system. All samples were analyzed for total plasma and total RBC LCFAs.

For the detection of total plasma oxylipins, we took a plasma sample (200 μL), added 300 μL of 10M sodium hydroxide (NaOH), and subjected it to alkaline hydrolysis at 60 °C for 30 min. The sample pH was then adjusted to 6 using 300 μL 58% acetic acid. The prepared samples were then subjected to solid phase extraction (SPE) using a Varian Bond Elut Certify II column. Specific experimental steps were described as previously [11 (link),45 (link)]. For the detection of free plasma oxylipins, SPE extraction was performed directly after pH adjustment without prior alkaline hydrolysis.
The extracted metabolites were evaluated by LC-MS/MS using an Agilent 6460 Triple Quad mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) and an Agilent 1200 high-performance liquid chromatography (HPLC) system (degasser, binary pump, well plate sampler, thermostatic column chamber). A Phenomenex Kinetex column (150 mm 2.1 mm, 2.6 m; Phenomenex, Aschaffenburg, Germany) was used in the HPLC system. The specific analysis process has been described previously [45 (link)]. Free and total plasma oxylipins were measured in the blood samples.