Washing, Elution and Reconstitution Optimization. Matrix effects were investigated in terms of SSE (%) and results for the two tested washing protocols were largely comparable (see Figure ). However, using two times 1 mL of H2O improved SSE by approximately 5% for most analytes, especially for those with RT between 1.0 min and 7.0 min (Figure ). Consequently, two washing cycles using 1 mL H2O each were used in the final protocol. The use of acidic MeOH solutions (3% FA) as elution solvent resulted in lower REs (<50% for most analytes), whereas better RE values (RE 50%-150% for most analytes) were achieved when eluting with pure MeOH or basic MeOH (3% NH3). Six non-polar compounds (LogP range of 4.0 -5.5) had surprisingly high RE values >150% using the basic MeOH solution while only one of the compounds had too high RE using pure MeOH (see Figure ). Therefore, pure MeOH was selected as the elution solvent for all further optimization steps. Furthermore, basic solvents and high concentrations of ammonia in the UPLC system may cause issues including unstable RTs and potential damage to both the UPLC column sorbent and the UPLC system itself . Subsequently, different elution solvents (MeOH, ACN and ISO) were compared and elution volumes were optimized. Recoveries of approximately 70% of the analytes were classified as "good" when using MeOH for elution, whereas this fraction reduced to 39-62% when ACN, ISO or a 1:1:1 mixture of all solvents was used (Figure ). Then, solvent volumes were optimized for MeOH and 200 µL, 400 µL and 800 µL were tested for elution (see Figure )). Approximately 70% of compounds achieved "good" REs when using 400 µL or 800 µL of MeOH for elution, whereas this fraction dropped to 63% when eluting with 200 µL MeOH. The smaller solvent volumes (200 µL) resulted in inefficient elution for around 10% of all analytes and 400 µL (using 2 times 200 µL) was selected for further method optimization steps, as it represented a well-balanced compromise between good RE and minimum solvent consumption. Regardless of the elution conditions, certain highly polar compounds in pooled urine were only poorly retained on SPE cartridges (i.e., with "poor" RE) and reversed-phase (RP-)LC columns (i.e., with RT <1 min using our LC method). As expected, primarily low-mass organic acids such as 5-hydroxymethyl-2-furanoic acid, dibromoacetic acid, dichloroacetic acid, bromoacetic acid, and p-hydroxybenzoic acid were only poorly retained on both SPE and RP-LC columns. The effect of drying the raw extracts followed by reconstitution was investigated (see Figure ), and diluting the raw extracts resulted in better ("good") recoveries performance, indicating the need to consider the optimization of reconstitution steps during method development. The lower percentage of analytes with "good" REs is clearly related to issues during resuspension, which might be caused by insufficiently short vortexing, the absence of a sonication step, or the selection of the resuspension solvent containing 10% ACN in H2O (1/1, v/v). Furthermore, a direct dilution approach is clearly favorable in terms of simplicity and speed, which are both required for high-throughput applications.