Agilent 1290 infinity 2 uplc system

A mixture of 190 µl of serum sample, 10 µl of internal standard (10 μg/ml, clenbuterol and chloramphenicol mixture) and 800 µl of methanol-acetonitrile (v/v = 1:1) solution was sonicated at 4°C for 10 min, then the mixture was incubated at –20°C for 1 h, followed by centrifugation at 13,000 g for 15 min at 4°C to obtain the supernatant. The supernatant was filtered by 0.22 µm microporous membrane. Finally, 3 µl of the filtrate-solution was transferred by an autosampler and injected into the UPLC-MS/MS system for metabolomic analysis. In addition, 10 µl of serum from each sample was mixed as a quality control (QC) sample. QC samples were processed in the same way as the study samples. Serum metabolomics analysis was performed with Agilent^® 1290 Infinity II UPLC system (Agilent Technologies Inc., United States) and AB Sciex^® Triple TOF 5600⁺ mass spectrometer system (AB Sciex, United States). UPLC-MS/MS analytical conditions used previous methods of our lab (Xu et al., 2021 (link)). In addition, QC samples were tested after every 10 samples in the analysis sequence to evaluate the reliability of the large-scale metabolomics analysis and the stability of the instrument (Dudka et al., 2020 (link); Zhu et al., 2021 (link)).

Components in PDBW were analyzed with a LC-MS/MS system. Chromatographic separation was performed using an Agilent 1,290 Infinity II UPLC system (Agilent, Santa Clara, CA, USA) equipped with an Agilent Eclipse XDB-C18 column (100 mm × 2.1 mm i.d., 3.5 μm). The column temperature was set at 35°C and the UV absorption wavelengths were set as 254 and 320 nm, respectively. Elution were accomplished on a gradient of formic acid (0.1%) in water (mobile phase A) vs. formic acid (0.1%) in acetonitrile (mobile phase B) at a flow rate of 0.3 mL/min and the injection volume was 10 μL. An optimal gradient elution program was applied to separate the components effectively: 0–15 min, 5–90% B; 15–20 min, 90% B. MS/MS analysis was operated using a high resolution mass spectrometer (Q-Exactive Focus, Thermo Fisher Scientific). The MS data were acquired from an electrospray ionization (ESI) source in positive and negative ion mode, respectively. The parameters of the source were set as follows: nebulizer gas pressure 45.00 psi; electrospray voltage 4,000 V; fragmentor 150 V; desolvation gas (nitrogen > 99.99%) flow 600 L/h; desolvation temperature 350°C and source temperature 100°C; target mass m/z 400; scan range m/z 100–1,500. Data acquisition processing was carried out using Thermo Fisher Xcalibur workstation (Xcalibur software, version 4.0).

The PGE₂, 5-HETE, 12-HETE, and 20-HETE quantification was performed on an Agilent Ultivo QQQ MS system coupled to an Agilent 1290 Infinity II UPLC system (Agilent, Santa Clara, CA, USA). Chromatographic separation of oxylipins was achieved using a gradient of water, methanol, and acetonitrile all with 0.1% acetic acid (v/v). Acquisition parameters were as previously described with minor modifications [20 (link)]. The acquired data were quantified by Quant-My-Way (Agilent, Santa Clara, CA, USA) using calibration curves.