Development of a quantitative method for the analysis of total l-ascorbic acid in foods by high-performance liquid chromatography

A new method has been developed for the determination of total vitamin C in foods. The method requires less time than the traditional methodologies and uses a radical oxidation of l-ascorbic acid (AA) to obtain dehydro-l-ascorbic acid (DHAA) by means of a peroxyl radical generated in situ by thermal decomposition of an azo-compound, 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH). The dehydro-l-ascorbic acid is condensed with benzene-1,2-diamine (o-phenylenediamine, OPDA) to form its highly fluorescent quinoxaline derivative, 3-(1,2-dihydroxyethyl)furo[3,4-b]quinoxaline-1-one (DFQ), which is then separated on a C18 column eluted with a mobile phase of 80 mM phosphate buffer and methanol at pH = 7.8 and detected fluorometrically at λex = 355 nm and λem = 425 nm. The reaction conditions for the complete conversion of AA to DFQ were 56 °C, 36 min and a μmol AAPH/AA ratio of 60. The sample, extracted with an aqueous metaphosphoric acid solution, was analyzed after being filtered through a 0.45 μm membrane. The method has shown good repeatability, sensitivity and accuracy compared to the results obtained with the reference method. The response of the detection system was linear within a range of 0.5–8.0 μg/mL with a correlation coefficient of 0.9997. The limit of detection was 0.27 μg/mL and the limit of quantification was 0.83 μg/mL. The AA contents of some selected foods were analyzed.