Thiol–Ene Click-Derived Porous-Layer Open-Tubular Column for Online Purification and Multiplex LC–MS/MS Analysis of Neurotransmitters

A thiol–ene-based porous stationary phase (PLOT-type coating) was developed for online sample cleanup and enrichment coupled to LC–MS/MS for the simultaneous determination of structurally diverse neurotransmitters in biological matrices. The resulting porous thiol–ene functionalized capillary column combines the inherently high permeability of the open-tubular architecture with tailored surface functionality, enabling direct injection and efficient handling of highly polar, low-abundance analytes without derivatization. The method allows multiplexed quantification of serotonin, dopamine, glutamic acid, γ-aminobutyric acid, and reduced glutathione in plasma, cell samples, and brain tissue within a simplified analytical workflow. Compared to conventional off-line protocols, the proposed approach minimizes sample preparation while maintaining high sensitivity and reproducibility, with recoveries ranging from 86% to 100% and matrix effects within 90–104%. The platform demonstrates reliable analytical performance across different biological matrices and is compatible with routine LC–MS/MS instrumentation. Overall, this work establishes the thiol–ene-porous capillary as a viable strategy for integrated sample processing in bioanalysis and expands its application to polar metabolite profiling in complex systems.