In optimization experiments, different eluent-column combinations were tested for the best overall performance and compatibility. Only eluents with a basic (pH 9.2) to slightly acid pH (pH 6) were combined with NH4F to avoid the formation of hydrofluoric acid. For a representative selection of selected compounds (25 metabolites on the HILIC column and 25 xenobiotics and human estrogen metabolites on the RP column), the averaged peak area (n=4-6) in a matrix-matched standard (urine) and a solvent standard both at a medium concentration level (level 6) were compared between the combinations (Figure and Table ). Peak areas were normalized to the best combination for each analyte to simplify the comparison. Endogenous metabolites are naturally, abundantly present in urine. Therefore the peak areas in the standard-spiked urine were increased compared to the solvent standards. The observed signal was mostly decreased for xenobiotics due to signal suppression in the urine matrix. The averaged peak areas of the selected metabolites showed that the combination of 2 mM NH4F/ACN on a SeQuant®ZIC®-pHILIC column (HILIC) and 0.3 mM NH4F/ACN on an Acquity HSS T3 is favourable (highest peak area) in the solvent. However, the difference between the three others is only minor, with combination 2 reaching about 85% of the average peak area of combination 1. In urine, the average peak area of combination 4, which uses an Acquity BEH amide column instead of a SeQuant ZIC-pHILIC column, is only about half of the others. Except for combination 4, all eluent-column combinations performed similarly, but with combinations 2 and 3, both using NH4F as an additive, up to four metabolites (choline, phosphocreatine, citric acid, 3methylcytidine) were not detectable at the chosen concentration level. Therefore the SeQuant ZIC pHILIC column with a basic NH4CO3 buffer based on seemed to be the best choice regarding endogenous metabolites excluding estrogen hormones. When xenobiotics and endogenous estrogen metabolites were investigated, however, the additive NH4F clearly outperformed the basic buffer. The average peak area in urine and solvent of this group nearly doubled when NH4F was used. Since xenobiotics are generally less concentrated than metabolites in real-life samples, we decided to use this additive to boost their sensitivity and finally selected the combination with 2 mM NH4F as aqueous HILIC eluent and 0.3 mM NH4F as aqueous RP column due to better overall performance.