Advanced applications of Diethanolamine-and Tris-derived mixed-mode stationary phases for separation of bioactive compounds and simultaneous determination of Sulphonamides in chicken eggs

Two mixed-mode stationary phases, DEA-Mix-SP and THMA-Mix-SP, were previously prepared, characterised, and tested with simple probes. In the present study, these columns were applied to the separation of biologically important and structurally diverse analytes. In HILIC mode, both phases resolved seven water-soluble vitamins and five biogenic amines, while RPLC mode enabled efficient separation of nine sulphonamides and twelve plant growth regulators including cytokinins, auxins, and chlorophenoxyacetic acids. Retention behaviour was also examined, supporting the understanding of analyte-phase interactions. Comparative studies with ACE C18 and aminopropyl silica confirmed the superior performance of the new phases, with THMA-Mix-SP achieving baseline separation of all nine sulphonamides and all five biogenic amines, demonstrating enhanced selectivity across analyte classes. Furthermore, DEA-Mix-SP was validated for quantitative determination of sulphonamide residues in eggs, providing excellent linearity, precision, and LODs of 0.01-0.075 mg/L. Residue analysis revealed sulfamethoxazole up to 4408 mu g/kg, far exceeding international zero-tolerance limits. These results confirm DEAMix-SP and THMA-Mix-SP as robust multifunctional platforms for bioanalytical separations and food-safety monitoring.