Amino Acid Quantification in the Presence of Sugars using HPLC and Pre-Column Derivatization with 3-MPA/OPA and FMOC-Cl

Determination of free amino acids (AA) in musts and young wines using pre-column derivatization with 3-MPA/OPA for primary AA and FMOC-Cl for secondary AA was studied. As samples differ in their sugar content considerably, the influence of sugars on derivatization was investigated using two internal standards: (i) primary AA norvaline and (ii) secondary AA sarcosine, in real samples and in model fructose and glucose solutions. A strong matrix effect was observed, especially when concentrations of sugars exceeded 50 g/L. The influence of sugars on derivatization of secondary AA sarcosine was more pronounced than on the primary AA norvaline. For quantitative evaluation of AA in samples with variable but high sugar content we propose to use external calibration in combination with internal standard method, where the response factors for individual AA are corrected with respect to that of internal standards.