Brief Workflow
Biological samples are homogenized and extracted using a modified Folch Method. Extracted samples are separated chromatographicaly on a Thermo Ultimate 3000 UHPLC system using an ACQUITY Premier BEH C8 Column (Waters, USA), and mobile phase consisting of water, acetonitrile, isopropanol, ammonium formate, and formic acid. The UPLC is coupled to a Q Exactive high-resolution accurate mass (HRAM) spectrometer (ThermoFisher Scientific, Waltham, MA). MS/MS spectral data is acquired with data dependent acquisition (DDA) method using Xcaliber software and generated spectral data is processed using MS DIAL following, based on both LipidBlast annotations and theoretical MS/MS spectra for lipids included internally in the software. Solvent blanks, method blanks, internal standards and pooled quality control (QC) samples are employed for quality assurance and quality control.
Detailed Workflow
Biological samples such as serum, milk, feces, tissue and/or cecum are homogenized using Precellys 24 Homogenizer. A portion of the homogenate is used to extract lipids using a modified Folch Method. Extracted samples are analyzed using Ultimate 3000 UHPLC system (ThermoFisher Scientific, Waltham, MA) system coupled to Q Exactive high-resolution accurate mass (HRAM) spectrometer (ThermoFisher Scientific, Waltham, MA). Chromatographic separations are performed on an ACQUITY Premier BEH C8 1.7µm x 2.1 x 100mm Column (Waters, USA) over the course of 35 minutes using a mobile phase system consisting of 60:40 water/acetonitrile (Solvent A) and 90:10 isopropanol/acetonitrile (solvent B), both containing 10mM of ammonium formate and 0.1% formic acid. MS/MS spectral data is acquired with data dependent acquisition (DDA) method in both positive and negative mode using Xcaliber (version 4.1.31) software. The DDA method consists of a full MS scan ranging from m/z 50 to 750, followed by MS/MS scans in the range from m/z 50 to 750, for the top N = 5. Solvent blank, method blank, internal standards and pooled quality control (QC) serum samples are employed for quality assurance and quality control of the lipid profiling. The generated spectral data is processed using MS DIAL (version 4.80) following the “lipidomics” method, based on both LipidBlast annotations and theoretical MS/MS spectra for lipids included internally in the software.