Polarity Gradient Solvent Confinement Membrane Cartridge\nto Broaden Metabolite Coverage of Plasma Untargeted Analysis

Various\npolarity chemicals exist in complex samples, such as plasma;\nnontargeted comprehensive analysis naturally requires multiple polar-extracted\nsolvents; consequently, the polarity of the solvent plays a crucial\nrole in the extraction efficiency of analytes from complex samples.\nIn the present study, based on the diffusion behavior and nanoconfinement\neffect of solvents in the nanoconfined space, the polarity gradient\nsolvent confinement liquid-phase nanoextraction (PGSC-NLPNE) protocol\naimed to perform a one-step nontargeted analysis of a wide range of\nmetabolites in plasma was established. The continuously wide range\nof extracted solvent polarities on carbon nanofibers/carbon fiber\n(CNFs/CF) membranes was achieved using a mixture of hexane, dichloromethane,\nmethanol, and water as nanoconfined solvents. The polarities (Log <i>P</i>) of gradient solvents ranged from −1.38 to 3.94.\nCorrelational analyses indicated that metabolites with Log <i>P</i> values ranging from −1.90 to 3.84 were closely\nrelated according to similarity-intermiscibility theory. Coupled with\na homemade modified guard column device, CNFs/CF membrane cartridge\n(CCMC), a PGSC-NLPNE-UHPLC-MS online protocol was established and\napplied in plasma untargeted analysis. By comparing metabolome coverage,\nreproducibility, and extraction recovery with protein precipitation\nand two-step liquid–liquid extraction commonly used in untargeted\nanalysis, the PGSC-NLPNE-CCMC protocol demonstrated higher reproducibility\nand recovery. This protocol has shown great potential for ultrafast\nanalysis of plasma untargeted metabolomics with broader metabolome\ncoverage. It could be a potential tool to rapidly screen out valuable\nbiomarkers related to diseases in the clinic.