A multi-criteria Gradient Score workflow for RP-HPLC-MS/MS method development: from column selection to segmented...

In this work, an extended Gradient Score workflow is proposed that integrates chromatographic performance metrics and MS/MS acquisition-related criteria into a unified multi-criteria evaluation framework for the development of targeted RP-HPLC-MS/MS method for profiling of a broad range of phenolic compounds in food matrices. The workflow comprised the selection of an appropriate reversed-phase stationary phase, followed by optimization of the gradient profile. Column performance was evaluated using complementary descriptors of critical-pair separation, peak width and analyte distribution, identifying the Raptor ARC-18 column as the most suitable stationary phase. Retention modeling based on linear solvent strength theory was subsequently employed to design segmented gradients for improved analyte distribution across the chromatogram. The results demonstrated that, compared with conventional linear gradients, optimized segmented gradient provided more favorable overall performance, combining improved analyte distribution, sufficient critical-pair separation, reduced scheduled MRM-window overlap, and acceptable analysis time. The developed method was finally applied to representative food samples, demonstrating its suitability for targeted profiling of chemically diverse phenolic compounds in complex matrices.