HPLC separation of biogenic amines using a C-12 hydroxy mixed-mode column: Investigation of the mixed-mode separation mechanism and simultaneous quantitative determination

This study presents the simultaneous quantification of biogenic amines in cheese samples using a C-12 hydroxy mixed-mode HPLC column. Histamine, dopamine, tyramine, serotonin, and tryptamine were analyzed without derivatization and detected with a UV detector at 254 nm. The mobile phase consisted of 50% acetonitrile and 50% sodium acetate buffer (25 mM, pH 5.5). The effects of pH, temperature, flow rate, and buffer concentration were evaluated. Under optimal conditions, all analytes were separated within 20 minutes. The performance of the C-12 hydroxy column was compared with a conventional C18 column and showed superior selectivity and sensitivity. The separation mechanism was examined using quantitative structure–retention relationship (QSRR) parameters such as logD, pKa, and polarity. The method proved effective for reliable determination of biogenic amines in various cheese samples.