Serum Metabolomics Profiling by UPLC-MS/MS

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)).