The Determination of Vitamin D in Fortified Milk and Other Foods by High Performance Liquid Chromatography(HPLC)
高效液相色谱法测定强化奶及食品中维生素D含量

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.