259 of the 4578 metabolite features in the discovery cohort (Fig. 1, step 2b) were selected as potential markers for PDAC as determined by the VIP-plot in the initial OPLS-DA model (Fig. 4a). 19 metabolites remained after feature quality control, detection in validation cohort and metabolite identification. These 19 metabolites were significant in the validation cohort OPLS-DA model (Fig. 4b). A comparison of the loading scatter plots for the discovery- and validation cohort OPLS-DA models showed that a group of 14 metabolites was regulated in opposite directions in the two cohorts (Fig. 4c, d). As in the univariate analysis, all 14 were phospholipids that were down-regulated in PDAC compared to CP in the discovery cohort but up-regulated in PDAC in the validation cohort.