One-step dispersive solid phase extraction (dSPE) and protein precipitation to streamline high-throughput reversed-phase metabolic phenotyping of blood samples

The chemical analysis of blood products (plasma and serum) is commonplace in metabolic phenotyping studies. The diversity of analytes in blood products characterized by varying physico-chemical properties, stability, and solubility presents an analytical challenge when attempting to achieve comprehensive analyte coverage by liquid chromatography mass spectrometry (LC-MS). While reversed-phase chromatography (RPC) of lipid analytes does not suffer from the presence of small molecules eluting mostly early in the chromatogram, the RPC-based analysis of low-molecular-weight metabolites (LMWMs) in minimally processed blood products is hindered by the presence of proteins and lipid species which fail to cleanly elute and negatively impact the assay. Here, we propose a novel application of dispersive solid phase extraction (dSPE) for the one-step single-phase depletion of proteins and lipids from plasma and serum samples without detrimental effect to the composition of LMWMs, overcoming challenges of conventional SPE. Using this approach, we demonstrate in two clinical studies the paired use of C18 RPC LC-MS for LMWM profiling enabled by dSPE and C8 RPC LC-MS for lipid profiling, providing 650+ annotated LMWMs and lipid species in plasma and serum samples.