A log P-charge dual-factor model-guided electromembrane extraction for high-throughput analysis of hydrophilic endogenous peptides in urine

Two different liquid membrane systems were developed for efficient extraction of hydrophilic peptides. • The mass transfer mechanisms for conventional organic and DES-based liquid membranes were systematically elucidated. • A high-throughput 96-well electromembrane extraction platform was established for urinary peptide analysis. • A novel Log P-charge dual-factor model was proposed to predict peptide electromigration behavior in EME systems. The selective enrichment of trace hydrophilic peptides from complex biological matrices remains a pivotal challenge in biomarker discovery. To address this, we developed a high-throughput and environmentally friendly electromembrane extraction-liquid chromatograph/mass spectrometry (EME-LC/MS) method for the analysis of 21 hydrophilic endogenous peptides in urine. Two different liquid membranes were optimized: P1, a conventional organic liquid membrane consisting of 2-nitrophenyl octyl ether (NPOE) and carvacrol (2:1, v /v ) with 2% di(2-ethylhexyl) phosphate (DEHP); and P2, a deep eutectic solvent (DES)-based membrane composed of camphor and decanoic acid (1:1, w /w ) with 0.5% DEHP. Mechanistic studies revealed that P1 extraction relies on cation-π, π-π stacking, and ion-pair interactions, enriching 13 peptides within 20 min at 30 V. In contrast, P2 operates via hydrogen bonding and ionic interactions, achieving more rapid extraction of 14 peptides within only 10 min at 30 V, along with a broader operational window and enhanced green solvent potential. Importantly, we established a log P -charge dual-factor model, demonstrating that peptide electromigration was co-determined by lipophilicity (log P ) and net charge under operational buffer, with its predictive reliability confirmed by external validation. Implemented in a 96-well format, the EME method was high-throughput, solvent-minimized, and environmentally friendly with AGREEprep score of 0.72. Beyond providing a robust analytical tool, this work established a fundamental mechanistic framework for the design of electromembrane extraction systems, advancing both biomarker analysis and the understanding of transmembrane transfer for polar biomolecules. Download: Download high-res image (101KB) Download: Download full-size image