Multiplex Isotope Tagging-Electromembrane Extraction of Metabolites with Diverse Polarities

Abstract In this work, a multiplex isotope tagging-electromembrane extraction (MIT-EME) strategy was proposed to address two critical drawbacks of conventional EME in metabolite analysis: failure to simultaneously extract polar and non-polar metabolites, and limited analytical throughput. Specifically, seven carnitines covering the hydrophobicity (log P) range of −0.5 to 3.2 were selected as model analytes, and were studied in human serum samples. The carnitines were labeled with p-dimethylaminophenacyl (DmPA) bromide and its two isotopic analogues for sample encoding. Labeled samples were then pooled and subjected to EME and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The labeling increased the lipophilicity of the carnitines (0.9 ≤ log P ≤ 4.6), thereby increasing the partition coefficient by more than two orders of magnitude and the transmembrane flux by up to 43.9-fold, ultimately enabling efficient simultaneous extraction of polar and non-polar carnitines with recoveries of 62−95%. The MIT-EME-LC-MS/MS method provided satisfactory limits of detection (0.02−0.09 ng mL−1), accuracy (90−113%), precision (≤14%), and matrix effects (90−107%). Benefiting from isotopic multiplexing, samples were pooled and analyzed in a single LC-MS/MS run, and achieved the synchronous analysis of three serum samples. The method was applied to serum samples from 100 hyperuricemia patients and 200 healthy individuals, and obvious intergroup differences of serum carnitines were observed. The strategy developed in this study provides a powerful approach for large-scale metabolomics analysis.