Untargeted metabolomics analysis is performed on solid and liquid matrices using a Thermo Vanquish Horizon UHPLC-coupled Exploris 480 Orbitrap high-resolution mass spectrometer following methanol extraction and centrifugation, with chromatographic separations via Waters Acquity Premier CSH C18 column and acquired spectra processed using Thermo Compound Discoverer 3.3. Identifications are based on matching against an in-house library comprising over 500 metabolites, retention time, MS/MS, and mzCloud (Thermo) and NIST spectral libraries. Box plot and PCA multivariate analysis are used to confirm centering, data normalization and quality.
Arkansas Children’s Nutrition Center (ACNC) Metabolomics and Analytical Chemistry Core Untargeted metabolomics analysis is performed on multiple matrices, including: foods and ingredients, biofluid – plasma/serum, urine, human milk, human and animal tissue, feces and cecal contents, cell culture media and cell fractions. Liquid samples are normalized by volume or osmolarity, spiked with labelled reference standards and extracted using a methanol “cold crash” and centrifugation, with solid tissues homogenized prior to extraction. Treatments and pooled quality control (QC) samples are analyzed using a Thermo Vanquish Horizon UHPLC-coupled Exploris 480 Orbitrap high-resolution mass spectrometer in both positive and negative electrospray ionization mode (ESI), with chromatographic separations via Waters Acquity Premier CSH C18 (1.7 µm x 2.1 x 100 mm) column and mobile phase gradient consisting of water and acetonitrile. Experimental pools, used for system equilibration and quality control (QC) are injected at a rate of 10% total injections. Data-dependent acquisition (DDA) and the Thermo AcquireX method are used to generate inclusion lists established from pooled samples and exclusion lists for filtering low abundance, irrelevant and redundant fragmentation ions following blank injections. Full scan resolution, 180,000 FWHM; AGC target, 5 x 106 ions; max injection time, 100 ms; scan range, 60-900 m/z; RF lens, 70%; intensity threshold, 1 x 104. ESI +/- data-dependent MS2 spectra generated from 4 consecutive injected QC pools for inclusion lists; and resolution, 30,000 FWHM; AGC target, 1 x 105 ions; max injection time, 50 ms; isolation window, 2 m/z; dynamic exclusion; 40 s and stepped HCD collision energy, 10, 20, 40; mass isolation width, 2 m/z for blank injection exclusion list. The acquired spectra and fragment profiles are processed using Compound Discoverer 3.3 with build-in identification, prediction, statistics and pathway analysis features. Normalized data is processed against a in-house spectral library comprising over 500 metabolites as well as two cloud-based spectral libraries – mzCloud (Thermo) and NIST high resolution spectral library. Box plot and PCA multivariate analysis are used to confirm data normalization, centering and evaluate data quality.