After abundance peak filtering, the discovery cohort data set contained 4578 metabolite features including isotopes, adducts and low quality features (Fig. 1). 254 of these features had a p-value < 0.05 as determined by Student’s t test with Welch’s correction. After (i) raw data examination, (ii) re-integration and confirmation of statistical significance, (iii) detection in validation cohort and (iv) feature identification, 17 metabolites remained. 11 of these metabolites had a p-value < 0.05 (Student’s t test) in the validation cohort as well. Fold-change calculations showed that the eight metabolites with the lowest intensity were down-regulated in PDAC compared to CP in the discovery cohort, as opposed to up-regulated in PDAC compared to CP in the validation cohort (Fig. 2). These eight metabolites were phospholipids. After exclusion of the phospholipids, the three metabolites glycocholic acid, hexanoylcarnitine and N-palmitoyl glutamic acid remained as single discriminative markers for PDAC compared to CP (Fig. 3). In the discovery cohort, applying an FDR cut-off of 5% to the 254 metabolites did not yield any significant metabolites. However the manual inspection and re-integration together with the validation in an independent patient cohort strengthens the findings.