A Vanquish Duo UHPLC system with two independent pumping systems and two different columns was used to optimize chromatographic separation for our highly diverse set of endogenous and exogenous analytes. Different column/eluent systems were examined. The RP gradient was derived from Jamnik et al. ( ) and the HILIC gradient was based on , and . The NH4F concentration in the aqueous RP eluent was increased to 0.6 mM NH4F to ensure at least 0.3 mM NH4F after dilution with the effluent from the other column before introduction to the MS. If NH4F was used in both eluents, the modifier's concentration was increased to in total 2 mM NH4F as used this concentration for the HILIC gradient. As a reverse-phase column (RP), an Acquity HSS T3 (1.8 μm, 100 x 2.1 mm) was used. For HILIC chromatography, two different hydrophilic interaction liquid chromatography columns (HILIC) were used, namely, a SeQuant®ZIC®-pHILIC (5 μm, polymeric, 150 x 2.1 mm) and an Acquity BEH Amide (1.8 μm, 100 x 2.1 mm). Eluent B was in all cases 100% ACN. The aqueous eluent (solvent A) was changed as stated in Table . The selection of the NH4F concentration in the aqueous HILIC eluent was based on at either 1 mM or 2 mM. The injection volume was 5 μL for all columns and experiments.