Although MeCN was the most efficient of the tested solvents for lipid and protein removal, its addition to samples containing high salt concentrations is known to result in inconsistent biphasic partitioning of metabolites, leading to poor reproducibility . A 1:1 combination of MeOH:MeCN provided a good balance in terms of adequate protein removal and high recovery of LMWMs (Figure .B), with the methanolic component also serving to eliminate biphasic partitioning. This result validates the observations from Southam and coworkers , who compared several monophasic and biphasic extraction methods for the analysis of LMWMs from plasma and urine samples by hydrophilic interaction chromatography (HILIC) and concluded that monophasic MeOH:MeCN 1:1 (v/v) was the optimal solvent composition in terms of reproducibility and number of detected putative metabolites.