Comparison of several HPLC methods for the analysis of vitamin C

Ascorbic acid is a water-soluble vitamin common in food and dietary supplements. A usual problem with ascorbic acid analysis is the lack of stability of its samples and standard solutions owing to oxidation. A procedure to protect ascorbic acid from oxidation using mercaptoethanol is described in this study in connection with the comparison of three HPLC measuring methods. Two reversed-phase columns were evaluated for the separation. One technique uses UV detection, and two others use MS/MS detection. The methods were calibrated for quantitation on different ranges of concentrations. The LC-UV method covers the range 3.9 μg/ml to 500 μg/ml, one LC-MS/MS the range 80 ng/ml to 20 μg/ml, and the other 0.1 ng/ml to 20 μg/ml. As a proof of functionality all three methods were utilized for measuring vitamin C in energy drinks and chews (gummies). The sensitivity of LC-MS/MS methods was not necessary for the analysis of those samples, but the high sensitivity can be beneficial for other types of sample such as environmental or biological, where the levels of ascorbic acid are very low. The study showed that the formation of 2,3-diketogulonic acid is not a likely path for ascorbic acid oxidation following hydrolysis as reported in some studies.