Targeted Metabolite Profiling in Plasma

We targeted metabolites commonly involved in previous studies, such as carboxylic acids and derivatives, fatty acyls, organooxygen compounds and steroids and steroid derivatives [20 (link)–22 (link)]. Using targeted high-throughput metabolomics, we reliably quantified 318 metabolites or metabolite ratios from plasma samples. For liquid chromatography – mass spectrometry (LC–MS) analysis, the samples were re-dissolved in 100 μL of acetonitrile/water (1:1, v/v) solvent and centrifuged at 14,000 g at 4 ℃ for 15 min. Then, the supernatant was injected. The analyses were performed using an UHPLC (1290 Infinity LC, Agilent Technologies) coupled to a QTRAP MS (6500 + , Sciex) in Shanghai Applied Protein Technology Co., Ltd. The analytes were separated on HILIC (Waters UPLC BEH Amide column, 2.1 mm × 100 mm, 1.7 μm) and C18 columns (Waters UPLC BEH C18-2.1 × 100 mm, 1.7 μm). Quality control (QC) samples were included in the sample queue to evaluate the stability and repeatability of the system.
MultiQuant or Analyst was used for quantitative data processing. The QCs were processed alongside the biological samples. Metabolites in the QCs with a coefficient of variation (CV) less than 30% were denoted as reproducible measurements [26 (link), 27 (link)].