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The resulting porous thiol\u2013ene 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, \u03b3-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\u2013104%. The platform demonstrates reliable analytical performance across different biological matrices and is compatible with routine LC\u2013MS\/MS instrumentation. 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