Xevo tq s mass spectrometry uplc ms ms

Plasma samples were prepared as previously described with minor modifications.^{30 (link)} Each 150 μL of cold organic mixture (ethanol/chloroform = 3:1, v/v) is used to extract small-molecule metabolites from 50 μL of blood sample, spiked with two internal standard solutions (10 μL of l-2-chlorophenyla-lanine in water, 0.3 mg/mL; 10 μL of heptadecanoic acid in methanol, 1 mg/mL). The sample extracts were centrifuged at 4 °C and 14,500 rpm for 20 min. The supernatant was split for lipid and AA profiling using Acquity ultraperformance liquid chromatography coupled to Xevo TQ-S mass spectrometry (UPLC–MS/MS, Waters Corp., Milford, MA) and for untargeted metabolic profiling using gas chromatography-time-of-flight mass spectrometry (GC–TOFMS).

The plasma samples were thawed and extracted with 3 vol cold organic mixture of ethanol and chloroform and centrifuged at 4 °C at 14,500 rpm for 20 min. The supernatant was split for lipid and AA profiling using Acquity ultraperformance LC coupled to Xevo TQ-S mass spectrometry (UPLC–MS/MS, Waters Corp., Milford, MA). Metabolic profiling of other metabolites including organic acids, carbohydrates, AAs, and nucleotides was done using Agilent 7890A gas chromatography coupled to Leco Pegasus time-of-flight mass spectrometry (Leco Corp., St Joseph, MI). The raw data files generated from LC–MS (targeted) and GC-MS (untargeted) were processed with TargetLynx Application Manager (Waters Corp., Milford, MA) and ChromaTOF software (Leco Corp., St Joseph, MI), respectively. Peak signal, mass spectral data, and retention times were obtained for each metabolite. The detected metabolites from GC–MS were annotated and combined using an automated mass spectral data processing software package.^{28 (link)} The levels of lipids and AAs detected from LC-MS were measured using the AbsoluteIDQ p180 Kit (Biocrates Life Sciences, Austria) commercially available. The reference standards of these measured lipids and AAs were integrated in the kit.^{29 (link)} More details of metabolomic experiments and data preprocessing are described in the following subsections.