A solvent QC sample containing endogenous metabolites at 1 µM (median of 186 ng/mL) and xenobiotics including human estrogens at approximately 100 ng/mL was injected throughout the sequence (n=11), spanning over a period of 40 h (Figure ). Only the most abundant ion species were considered. The peaks from the HILIC column were evaluated for assessing endogenous metabolites and the peaks from the reversed-phase column for evaluating xenobiotics and human estrogens.At the chosen concentration level, 113 out of the 146 metabolites (77%) retained with a retention time bigger than 1 min on the HILIC column and 104 out of the 106 xenobiotics and human estrogens (98%) with retention on the RP column (retention time > 0.8 min) were detectable. Four analytes were not detectable even at higher levels. Therefore the best ionization mode remains unclear. The relative standard deviation (RSD) was calculated for the peak area and the retention of all detectable molecules at this level (Figures and). The median RSD of the area was 21.7% and 31% in negative and positive ionization mode, respectively. The differencebetween peaks from the HILIC and RP separation was minor, but in positive ionization mode, the relative standard deviation of the area was generally bigger. Regarding retention time, the median RSD was 0.4% for negative mode and 0.7% for positive mode. The 'xenobiotics' 2) . The best combination is shaded in grey. Different columns, namely Acquity HSS T3 (HSS), SeQuant®ZIC®-pHILIC (ZIC), Acquity BEH Amide (BEH), and eluents, namely 0.3/0.6 /1/2 NH4F in H2O (0.3/0.6/1/2 mM NH4F), 10 mM NH4HCO3 (pH 9.2) in H2O/ACN (9:1, v:v) (10 mM NH4HCO3), 50 mM CH3COONH4 (pH 6) in H2O (50 mM HAc) were tested times were more stable with a median of 0.3% compared to 1.3% for the endogenous metabolites. The retention time of some endogenous phosphates and acids, analysed mainly in negative ionization mode, was shifting significantly during the sequence as the buffer capacity of the NH4F eluent was low. The variation of the retention time and area of the most abundant ion specias for the respective analytes is also presented in quality control charts (Figure ). Pooled quality control samples of both sample sets were measured several times throughout the measurement (Figure ). The relative standard deviation of the retention time and the Figure Repeatability and accuracy in a solvent QC sample (1 µM for metabolites and xenobiotics at approximately 100 ng/mL) are reported as the number of analytes within a certain relative standard deviation of A) peak area and B) retention time. The lines mark the thresholds and the labels indicate how many analytes fall below a respective RSD percentage. Only the most abundant ion species was considered in the respective column. C) Analytes reported in SRM3672 and D) in SRM1950 with the grey line indicating the certified reference value and the expanded uncertainty according to the certificate of analysis normalized peak area was calculated for highly diverse analytes. This included bisphenol A (BPA), daidzein, enterodiol, glycitein, methylparaben and triclosan in the pooled urine of the Nigerian samples (n=9) and BPA, daidzein, nonylphenol and phydroxybenzoic acid in the pooled urine of the Austrian samples (n=5). In addition, four metabolites (tryptophan, uracil, fumaric acid, gluconate) were investigated. The relative standard deviation of the retention time was < 1.5% for all analytes except fumaric acid (about 2.8%). The relative standard deviation of the normalized area was < 20% for all six analytes except for BPA (22%), which was only at a concentration around the LOD in the Austrian pooled urine sample. The variation tended to be lower if the corresponding 13 C-labelled compound was available for compound-specific internal standardization (e.g. for methylparaben) as compared to surrogate internal standardization.