Optimization of an online post-column derivatization system for ultra high-performance liquid chromatography (UHPLC) and its applications to analysis of biogenic amines.

We investigated the efficacy and utility of a post-column derivatization method for the detection of amines with o-phthalaldehyde (OPA) in ultra high-performance liquid chromatography (UHPLC). Since it was anticipated that a derivatization reaction system placed downstream of the column would give considerable band broadening to narrow peaks generated by a UHPLC column, we examined the contributions of the dimensions of the reaction tube, the fluorescence (FL) detector flow cell, and the column to the extra-column peak broadening, and optimized the post-column derivatization system in UHPLC. We successfully separated amines within 6 min with gradient elution by using the post-column derivatization system connected to a UHPLC system. The analysis time was reduced by a factor of 7 compared with that by conventional HPLC. The detection limits were 18.0 - 105.7 fmol, depending on the compounds; the reproducibilities were 0.11 - 0.82% RSD for the retention times and 0.78 - 1.66% RSD for the peak areas (n = 10), respectively. The linear dynamic ranges were up to 12, 20, and 40 pmol/μL, depending on the compounds, respectively.