Development of a highly sensitive, highly selective and high-throughput method for determination of total ascorbic acid

A highly sensitive, highly selective, and high-throughput method for determination of intracellular total ascorbic acid (AA) was developed by measuring the fluorescence intensity of 1,2-diamino-4,5-methylenedioxibenzene (MDB)-labeled AA. First, the reaction buffer, temperature, pH, and time were investigated to establish optimal reaction conditions, and it was found that AA was detected with high sensitivity by reacting oxidized AA and MDB in 100 mM citrate buffer (pH 5.0) for 10 min at room temperature. Next, a scale-up synthesis was performed to determine the chemical structure of the MDB-labeled AA, and its excitation and emission wavelengths were optimized to 375 and 450 nm, respectively. HPLC analysis revealed that MDB reacts with AA with high selectivity under the established conditions. It was also found that AA could be determined under optimal reaction conditions, regardless of the presence or absence of cellular components. In conclusion, the established method for determination of AA with MDB can quantify AA in the range of 25 nM to 20 μM in a microplate with a detection limit of 10 nM. Furthermore, the method was confirmed to be able to quantify intracellular AA in a cell line by comparison with a standard HPLC method. This method is applicable to highly sensitive, highly selective, and high-throughput analysis and could be applied to the determination of AA in various cells used in the evaluation of AA activity.