The determination of vitamin D in fortified milk and other foods by high performance liquid chromatography (HPLC)
Wu, H.; Cheng, H.; Tian, J.; Wang, X.
Se Pu 15(1): 43-45
An HPLC procedure for determination of vitamin D(VD) in fortified milk and food has been developed. The samples were saponified by adding KOH, ethanol and vitamin C at 75 degrees C for 30 min. The digest was extracted with hexane in multistep manner. The extract was washed with 5% KOH, distilled water and 55% ethanol, and evaporated under reduced pressure at 50 degrees C. The residue was dissolved in hexane, transferred to a coentrifuge tube, evaporated under N2 and then dissolved in 200 microL mobile phase. The latter was injected into a silica column (10 microm, 30 cm x 4 mm) for cleanup, eluted with mixture of cyclohexane-hexane containing 0.8% isopropanol and detected at UV 254nm. VD fraction was collected according to the retention time of standard, evaporized under N2, redissolved in 100 microL MeOH, and separated on a C18 column (10 microm, 30 cm x 4 mm) with MeOH/H2O (98:2) as eluent. VD2 and VD3 were eluted as a mixture under the conditions. Recoveries of the method were 94.8% to 99.7%. The average CV (n = 5) for within and between day measurements were 1.6% and 2.1% respectively. This method was already used to determine the content of vitamin D in milk powder, pulvis elementi compound, mixed proteins, dried meat floss, humanized milk power, children health drink, milk wheat powder and fortified milk. The method is simple, rapid and accurate.