Fat Soluble Vitamins in Dietary Supplements: Optimization of Extraction, Simultaneous Determination by HPLC And Stability Studies
In recent decades, consumption of multivitamin/mineral (MVM) dietary supplements has significantly increased, reflecting a huge demand from the commercial market. Meanwhile, there is a big concern of the stability and shelf life of vitamins, including fat soluble vitamins (FSVs) in the dietary supplements. Therefore, it is necessary to conduct precise analysis and stability evaluation of the FSVs in the commercial products so as to scrutinize their content levels. An optimized method to extract the encapsulated FSVs (i.e. vitamin A acetate, vitamin A palmitate, vitamin D3 and vitamin E succinate) from MVM supplements was developed in light of four critical factors, including the hydrolysis time by HCl solution, concentration of HCl solution and hydrolysis temperature, as well as the extraction solvents. Based on the response surface methodology (RSM) the extraction yields of the FSVs were fitted to second order polynomial models, for which an optimized extraction condition was adopted with the following optimal parameters: 0.4 N HCl solution was used for MVM tablet samples at 50degC for 2 min. In addition, a mixture of hexane:ethyl acetate (7:3, v/v) was found to be the best solvent for an efficient extraction of the FSVs from the MVM supplement. Under this condition, extraction recoveries of the vitamin A esters (i.e. retinyl acetate and retinyl palmitate) were determined in a range from 92% to 106% and those for the vitamin E succinate and vitamin D ranged from 78% to 90% Subsequently, a chromatographic method for simultaneous determination of the aforementioned four FSVs by high performance liquid chromatography coupled with diode array detector was developed and validated in terms of high analytical precision and accuracy. For the four FSVs, the analytical limit of detection and limit of quantitation were determined to be in the range of 0.52 3.28 ug/mL and 1.56 9.94 ug/mL, respectively. The intra day and inter day tests for the analytical precision in terms of their relative standard deviations were determined to be below 7% for all of the four FSVs. In order to evaluate the stability of encapsulated FSVs, effects of light, temperature, pH, and storage conditions on the retention of FSVs were investigated. When dispersed in water and exposed to light, vitamin A acetate retained only 10% of its original amount after one day of storage, compared to its retention at 72% stored in darkness. In comparison, vitamin D3 and vitamin E were more stable than the vitamin A esters under the light exposure. Vitamin D3 and vitamin E possessed high retentions (72% and 50% respectively) when they were exposed to light after 5 days, and even higher values at 78% and 84% when they were in darkness during the same period. On the other hand, all four FSVs have shown significantly higher degradations at 30degC, 40degC, and 60degC than at 25degC in course of the thermal treatment for five consecutive days, but all of their degradation rates were determined to follow the first order kinetic models.