Where LC-MS is employed, RPC has long been the staple of metabolomics methodology providing an exceptionally broad coverage of the metabolome 3 through a combination of more hydrophobic interactions of analytes with alkyl chains of RP column and more hydrophilic interactions with residual silanols. RPC is especially well suited for the analysis of moderately hydrophobic endogenous molecules and much of the exposome: dietary metabolites , xenobiotics , synthetic chemicals , and microbiome products . However, direct application of RPC in the analysis of human blood products is challenging in practice due to the abundance of protein and complex lipids such as glycerophospholipids and triacylglycerols. Proteins are removed by organic solvent precipitation allowing their separation by centrifugation or filtering from the small molecule-containing supernatant. Lipids are strongly retained under RPC conditions and highly ionizable in electrospray ionization (ESI). Consequently, complex and neutral lipid species can accumulate in the LC system during analyses of low-molecular-weight metabolites (LMWMs) in blood products, eluting unpredictably from the column and exhibiting suppressive effects on the ionization of other metabolites . To better accommodate lipids in profiling analyses, the established standard is to design methods which measure LMWMs and cleanly elute lipids either simply preventing their problematic accumulation or allowing their additional measurement. The latter may be impractical due to the differences in solubilization of LMWMs and hydrophobic lipids in the sample diluent. Independent lipidomics and metabolomics analyses for one sample require separation of lipids and LMWMs using various sample preparation strategies. The gold standard here is liquid-liquid extraction (LLE) using different organic solvent mixtures allowing to achieve biphasic separation with lipids partitioned into the organic phase and LMWMs into the aqueous phase . To avoid the use of harsh organic solvents (e.g. chloroform in Folch and Bligh-Dyer methods), methyl tert-butyl ether (MTBE) was used by Matyash in 2008 allowing a phase distribution with the upper layer being the organic solvent (lower density MTBE) rich in lipid species, the lower aqueous layer rich in LMWMs, and the protein pellet collected at the bottom of the vial. However, sample extraction using two immiscible solvents has the risk of metabolites being incompletely partitioned between the two phases. It has been shown that lysophospholipids (LPC and LPE) as well as some fatty acids were highly recoverable in Matyash and Bligh-Dyer aqueous phase, and that some LMWMs, such as acylcarnitines, were recovered in both organic and aqueous phases of Matyash method . It has also been suggested that LMWMs were lost in biphasic extraction by all Folch, Bligh-Dyer, and Matyash methods , and some lipid classes (especially more polar phospholipids) were not extracted quantitatively . This emphasizes the need for more efficient, safe, easy, and reproducible serum and plasma content separation to achieve a dramatic reduction in lipids abundance required for sustainable RPC metabolic profiling of LMWMs.