Washing, Elution and Reconstitution Optimization. Thereafter, washing, elution and reconstitution steps were optimized. Matrix effects were investigated in terms of SSE (%) and results for the two tested washing protocols were largely comparable (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 RE (<50% for most analytes), whereas better RE (50%-150% for most analytes) was achieved using pure MeOH or basic MeOH (3% NH3) for elution. Six nonpolar compounds (LogP, 4.0 -5.5) had surprisingly high RE values >150% using the basic MeOH solution while only a single compound had RE>150% using pure MeOH (Figure ). Thus, pure MeOH was selected as the elution solvent for further optimization. In addition, it should be noted that basic solvents with high ammonia concentrations may cause issues including unstable RTs and potential damage to both the UPLC column sorbent and the UPLC system itself . Different elution solvents (MeOH, ACN and ISO) were compared and elution volumes were optimized. "Good" REs were achieved for approximately 70% using MeOH for elution, whereas this reduced to 39-62% when ACN, ISO or a 1:1:1 mixture of all solvents was used (Figure ). 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, as it represented a well-balanced compromise between good RE and low solvent consumption. Regardless of the elution conditions, certain highly polar compounds in pooled urine were poorly retained on the SPE cartridges. These compounds were primarily low-mass organic acids such as 5-hydroxymethyl-2-furanoic acid, dibromoacetic acid, dichloroacetic acid, bromoacetic acid, and p-hydroxybenzoic acid and were only weakly retained by the reversed-phase (RP-)LC column and with the used method (i.e., RT <1 min). The effect of drying the raw extracts followed by reconstitution was investigated (see Figure ), and diluting the raw extracts resulted in improved recoveries, indicating the need to consider the optimization of reconstitution steps during method development. Approximately 6% of analytes fell into the "acceptable" RE category when using drying and reconstitution, compared to the dilution method. Potential causes include short vortexing, the absence of sonication steps, or the selection of the resuspension solvent containing 10% ACN in H2O (1/1, v/v). For example, the RE for 2tert-butylphenol decreased significantly from 99.7% with dilution to 23% after drying and reconstitution. Furthermore, the study found an increased background signal for certain plasticizers, such as bisphenol F and S, when using drying and reconstitution. Consequently, a direct dilution approach is clearly favorable in terms of simplicity and speed, which are both required for high-throughput applications.