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Nutrition for Optimal Wellness

Accurate Analysis of Vitamin C in Dietary Supplements

NOW Foods Quality Department
Vitamin C is a water soluble vitamin required for several functions in our body. It is also known for its potential as an antioxidant and a stimulator for collagen production. In a recent study on various antioxidants including Vitamin E, Olive fruit extract, Grape seed and Green tea extracts, Vitamin C showed highest potential to maintain plasma antioxidant reserve.However, from quality control and analytical standpoint, it is very unstable and it is very challenging to accurately and correctly determine the amount of Vitamin C in a dietary supplement. The scientific team at NOW Foods has developed and validated a method to determine Vitamin C under standardized conditions that make this quantitation precise, accurate, and reproducible.
The main biologically active form of Vitamin C is Ascorbic acid (AA). AA is reversibly oxidized to form Dehydroascorbic acid (DHA), which also exhibits biological activity.  DHA is further oxidized to form diketogulonic acid, which has no biological function2. Since DHA can be easily converted into AA in the human body, the Vitamin C activity should measure both AA and DHA.3 We measure both Ascorbic Acid (AA) and (DHA) to determine Vitamin C activity. Our results have shown that the DHA is present in very small to negligible amounts in our dietary supplements. This concludes that majority of Vitamin C is present in the reduced (non-oxidized) form and demonstrates the high quality of the NOW supplements.
Vitamin C can be easily oxidized by temperature, pH, oxygen, light and contact with traces of copper and iron.  Both forms (AA and DHA) are present in the samples in a delicate equilibrium, their quantity depending upon the sample background and shelf life history. The objective of an analysis is to analyze both AA and DHA without disturbing the equilibrium between the two forms in the sample. The samples are dissolved in extraction solutions designed to stabilize AA and DHA and stored under refrigeration to prevent degradation. Samples are prepared under subdued lights. In a stability study conducted in our laboratory, three concentrations of Vitamin C solutions were analyzed at regular intervals. Our tests showed that under the testing conditions, AA was 100% stable up to at least five hours with in the experimental error of <2%.  Sample solutions are analyzed within five hours of preparation.
Several analytical methods have been used for the determination of Vitamin C. We can categorize them into two groups: Non-Separation and Separation Methods.
In the Non-Separation method the Vitamin C is analyzed in the presence of all other components of the supplement. Separation methods include methods where vitamin C is separated from other components in a formula. Most of the non-separation techniques give overestimates, due to the presence of oxidizable species other than AA. For example, the AOAC Official Method5 is a titration method that is complete when a pink color appears. The method is based on the oxidation of Vitamin C by a dye. Other natural components present in the sample (like tannins, betanins, copper, iron and manganese) can also be oxidized by the dye and thus contribute to higher results and low accuracy and reproducibility.
At NOW, we use a High Pressure Liquid Chromatography (HPLC) technique, based on the separation principle, where AA and DHA are separated from the rest of the components of the sample in an analytical column4 and quantified based on their absorption values in relation to standard ascorbic acid absorption. This technique provides the highest level of accuracy and reproducibility. The calibration curve of AA is linear over a range of 10 to 60 mg /ml with R2 =0.9999 and relative standard deviation of 0.2%.
DHA is easily separated from AA with the separation techniques described above, however, unlike AA it does not have enough absorbance value to be determined directly. DHA is therefore determined by a subtraction method. In this method, the samples are first analyzed for AA by HPLC assay, while a duplicate sample is treated using a special process. This treatment converts all DHA in the sample to AA, after which the sample is then analyzed by HPLC for AA. DHA is then calculated as the difference between the total AA after the DHA was converted to AA versus the AA content of the original sample. 
The analysis of Vitamin C potency in dietary supplements must include individual determination of AA and DHA by HPLC assays. Due to the highly unstable nature of both AA and DHA, assays must be performed under conditions that ensure their stability during analysis. The method used at NOW includes dissolving samples in refrigerated extraction solvent, diluting samples in refrigerated buffer material, keeping samples in a refrigerated autosampler until injection on the HPLC column. This method has been effective in providing accurate, reliable and reproducible results, controlling the interlaboratory discrepancies and has been successfully implemented at the NOW Foods analytical laboratory.
1  Rabovsky,A., Cuomo,J. & Eich,N. Measurement of plasma antioxidant reserve after supplementation with various antioxidants in healthy subjects. Clinica Chimica Acta 371 (2006) 55-60.
2  Deutsch, J.C. Dehydroascorbic Acid. J. Chromatography A, 881 (2000), 299-307.
3  Lee,S.K., & Kader, A.A. Preharvest and postharvest factors influencing vitamin C content of horticultural crops. Post harvest Biology and Technology, 20 (2000), 207-220.
4  Hernandez,Y., Lobo, G.M., Gonzalez, M. Determination of Vitamin C in tropical fruits: A comparative evaluation of methods. Food Chemistry 96 (2006) 654-664.
5  AOAC (1990). Official methods of analysis of the Association of Official Analytical Chemists, 15th ed. (pp1058-1059), Arlington VA:Association of Official Analytical Chemists.