Next, other extraction parameters were considered, namely liquid membrane volume, sample pH, acceptor pH, agitation rate, extraction voltage, and time. To improve the kinetics of the extraction, smaller sample and acceptor vials (200 µL / 100 µL, total capacity / working volume) were used during optimization. For both A1 and A2, 9-10 µL of liquid membrane was optimal, while smaller volumes gave rise to greater variability between replicates (Figure ); 9 µL was therefore selected for both methods. The effect of sample pH was investigated for pH 7.4, 9.0, and 11.0. Increasing the sample pH may theoretically improve the extractability for weaker acids, however, all model analytes were already negatively charged at pH 7.4. As such, pH 7.4 was optimal in both systems (Figure ). The decreasing performance at higher pH is explained by electrostatic repulsion of anionic analytes by negatively charged thymol (pKa 10.6) molecules in the sample-liquid membrane interface. Sample pH 7.4 was adjusted by diluting the plasma sample 1:1 (v/v) with 50 mM phosphate buffer (pH 7.4). The acceptor pH was investigated in the range pH 7.0-11.0 (S7-8), and found optimal at pH 10 in both systems. pH 10.0 was therefore selected for the acceptor, which was adjusted using LC-MS compatible (volatile) 50 mM NH4HCO3 buffer. The difference in optimal sample and acceptor pH is explained by the formation of boundary layers at the waterliquid membrane interfaces that locally have a higher and lower pH than the bulk aqueous solution, at the cathode and anode-side of the liquid membrane, respectively . For EME of acids, the acceptor-side boundary layer is thus more acidic and requires a higher acceptor pH to be overcome. Agitation rate was studied at 500-1000 RPM (Figure ), with 750 RPM found as optimal in both systems. This is concurrent with previous findings in the commercial extraction format . Finally, the impact of extraction voltage and time was investigated. The results are summarized by Figure as continuous boxplots of recoveries of all analytes extracted with acceptable efficiency (R > 40%). Based on these, 30 V and 10 V was selected for system A1 and A2, respectively, as only minor gains were made at greater voltages. Additionally, greater robustness is expected by operating the systems below the maximum tolerable voltage. Optimal extraction time was set to 30 minutes and 20 minutes for system A1 and A2, respectively, as a compromise between extraction efficiency and throughput. The final optimized parameter settings are provided in Table .