Metabolite Identification and Quantification Workflow

Following metabolite identification, all data were processed using the TargetLynx application manager for MassLynx 4.1 software (Waters Corp.). A set of predefined retention time mass-to-charge ratio pairs corresponding to metabolites included in the analysis was fed into the program. Associated extracted ion chromatograms (mass tolerance window = 0.05 Da) were then peak-detected and noise-reduced in both the LC and MS domains such that only true metabolite-related features were processed by the software. A list of chromatographic peak areas was then generated for each sample injection. An approximated linear detection range was defined for each identified metabolite, assuming similar detector response levels for all metabolites belonging to a given chemical class represented by a single standard compound (35 (link)). Metabolites for which more than 30% of data points were found outside their corresponding linear detection range were excluded from statistical analyses.
Normalization factors were calculated for each metabolite by dividing their intensities in each sample by the recorded intensity of an appropriate internal standard in that same sample, following the procedure described before (36 (link)).