Method Development and Optimization. The yield of liquid-liquid extraction depends on the partitioning of the compound between two immiscible solvents, i.e., the extraction solvent and the sample matrix. EC is a polar molecule and soluble in most solvents with medium polarity such as ethers to polar solvents such as water. Its partitioning between ethyl acetate and water is nearly 1:1 based on our preliminary data. The extraction yield of EC increased with the ratio of solvent-to-plasma and plateaued when the ratio was 4:1 or higher (Figure ). Ethyl acetate, a more polar solvent than MTBE, gave a higher recovery than MTBE, which agrees to the finding of Xu et al . who compared ethyl acetate with diethyl ether and methylene chloride. Methylene chloride was not considered in this study due to its toxicity. Ethyl acetate was chosen for the extraction of EC in plasma. MTBE was used for the extraction of distillers grains samples because of its faster evaporation rate and the high levels of EC in distillers grains co-products. A 32% increase of extraction yield was observed when NaCl was added to the deproteined plasma (Figure ). Although the presence of plasma proteins showed no impact on the EC recovery, in order to utilize the salting-out effect, plasma proteins would first need to be removed. Otherwise, upon mixing, a stable emulsion would form after NaCl was added. The same emulsion issue also occurred during hexane wash if the proteins were not previously removed. Hexane wash aimed to remove the neutral lipids which would be otherwise extracted with EC into the organic solvent and injected into the GC. The non-volatile nature of neutral lipids would shorten the GC column lifetime and requires more frequent cleaning of the GCMS system. AOAC 994.07 and Nóbrega et al. stated that precaution is needed to avoid complete solvent removal of the EC extract. Other reports specified a final solvent volume of the extract after evaporation . These notes suggested that EC is recovered poorly after the solvent is completely removed. The recovery of EC during solvent evaporation was examined in the present study and no loss of EC was found during evaporation under nitrogen at ambient temperature for up to 2 h (Figure ). However, as soon as the solvent was depleted, EC would be lost, presumably due to evaporation.