UHPLC-QTOF/MS analysis was performed using a 1290 ultra-high performance liquid chromatography system (Agilent, California, United States) and a 6540 Q-TOF mass spectrometer (Agilent, California, United States), operated in positive and negative ion modes. MS parameter settings are shown in Supplementary Table S2 . Analysis were separated on a Waters BEH C18 column (2.1 × 150 mm, 1.7 μm, Milford, MA, United States) at a constant flow rate of 0.4 ml/min at a column temperature of 50°C. LC conditions were as follows: 0.1% formic acid in deionized water (solvent A) and 0.1% formic acid in acetonitrile (solvent B); gradient elution method is shown in Supplementary Table S3 .
1290 ultra high performance liquid chromatography system
Manufactured by Agilent Technologies
Sourced in United States
The 1290 ultra-high performance liquid chromatography system is a high-performance liquid chromatography (HPLC) instrument designed for advanced analytical separation and detection of chemical compounds. It features a compact design, precise temperature control, and advanced data acquisition capabilities to enable efficient and reliable analysis of complex samples.
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2 protocols using 1290 ultra high performance liquid chromatography system
1
UHPLC-QTOF/MS Analysis of Metabolites
2
Untargeted Metabolomics Using NGMS
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Untargeted metabolomics was performed following our previously published NGMS approach. 4 (link) In brief, analyses were performed using a 1290 ultra-highperformance liquid chromatography system (Agilent) coupled to a 6545 QTOF mass spectrometer (Agilent) equipped with a dual electrospray ionization source. Each sample was run in duplicate in both positive and negative ionization modes. A 2.0-μL aliquot of extracted plasma sample was injected onto an Acquity UHPLC HSS T3 (C18, 2.1 × 100 mm, 1.8 μm) column (Waters) operating at 40 • C. Chromatographic separations were performed by applying a binary mobile phase system. The buffer composition and mass spectrometry settings used were as previously reported by Coene et al. 4 (link) For the analysis of the NGMS data, alignment, annotation, and statistical testing were performed in an integrated manner through an in-house bioinformatic pipeline. 14 (link) Both the analytical and data analysis workflow for NGMS data were validated for clinical diagnostic use according to ISO15189 standards.
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