Bile acid extraction was performed by adding methanol containing internal standards to 50 μL plasma. Samples were analyzed using an ACQUITY UPLC system with an HSS T3 column coupled to a 6530 Accurate Mass QToF (Agilent Technologies).
Following liquid chromatography‐mass spectrometry analysis, data was preprocessed by performing peak integration, background correction, and determination of the relative ratios between metabolites and their corresponding internal standards. The metabolite units were expressed as the peak area ratios of the target analyte to the respective internal standard. An in‐house written tool was applied using the QC samples to compensate for shifts in the sensitivity of the mass spectrometer throughout the batches. Both internal standard correction and QC correction were applied to the dataset before reporting results. Quality assurance of metabolite measurements was performed using the QC relative standard deviation (RSDqc). For amines, reported compounds had an RSDqc <15%, for positive and negative lipids RSDqc <20%, and for bile acids RSDqc <30%.
Following liquid chromatography‐mass spectrometry analysis, data was preprocessed by performing peak integration, background correction, and determination of the relative ratios between metabolites and their corresponding internal standards. The metabolite units were expressed as the peak area ratios of the target analyte to the respective internal standard. An in‐house written tool was applied using the QC samples to compensate for shifts in the sensitivity of the mass spectrometer throughout the batches. Both internal standard correction and QC correction were applied to the dataset before reporting results. Quality assurance of metabolite measurements was performed using the QC relative standard deviation (RSDqc). For amines, reported compounds had an RSDqc <15%, for positive and negative lipids RSDqc <20%, and for bile acids RSDqc <30%.