Preparation And Relative Correction Factors Study Of Vitamin D₃ Impurities

Vitamin D3 is a kind of fat-soluble vitamin,which plays an important role in the maintenance of organic systems.With the population aging and the development of animal husbandry,the demand of Vitamin D3 will keep rising.Nowadays,Vitamin D3 is mainly produced using cholesterol as starting material by traditional chemical route including esterification,bromination,elimination,hydrolysis and ultraviolet irradiation.In some methods bromination step was replaced by oxidation.However,the specificity of these methods is not high enough resulting in a major product and some other undesired byproducts such as trans-vitamin D3,provitamin D3,lumisterol3,iso-tachysterol3,tachysterol3.According to the requirements of the European Pharmacopoeia the amount of trans-vitamin D3,provitamin D3,lumisterol3,iso-tachysterol3,tachysterol3 should be less than 0.1%respectively(EP8.0).Therefore,it is necessary to monitor the amount of main component as well as the impurities during vitamin D3 producing process.The European Pharmacopoeia recommends HPLC-UV method for the quantification of various impurities by area normalization method which is not accurate enough since the UV response of various impurities at 265nm are not the same.Accurate quantification of related compounds in vitamin D3 is often hampered by the lack of commercially available standards.In this work,three impurities including trans-vitamin D3,lumisterol3,tachysterol3 were purified(purity>97%)with semi-preparative HPLC and identified by LC-MS and NMR.The UV relative correction factors(RCFs)at 254,265,272,281 nm for trans-vitamin D3,lumisterol3,tachysterol3 to vitamin D3 were measured.They are 0.447,0.614,0.729,0.909 for trans-vitamin D3,0.136,0.208,0.269,0.350 for L3,0.376,0.635,0.894,1.465 for T3 respectively.Then an accurate quantitative method for simultaneously determining vitamin D3 and the impurities in vitamin D3 using one reference substance was established.The contents of vitamin D3 were determined by the external standard method and the contents of the other three impurities were calculated according to their RCFs.Results show this method has high reliability and could be used in quality control of vitamin D3.In a successful fortification program,the stability of micronutrients added to a food is an important factor.In terms of vitaminD3 fortification,the search for any degradation products in fortified food,most notably in dairy products,can result in the discovery of the mechanism of vitamin D3 degradation which can play an important role to minimize it.Determination of vitamin D3 and its degradation products has been a challenge due to the low amount of their contents even in vitamin D3 enriched products.However,the information on compounds related to vitamin D3 degradation products in foods is limited.In this work,the stability of vitamin D3 at 40℃,60℃,80℃,100℃ in air for different time was investigated.Degradation products were preliminarily analyzed by HPLC,HPLC-MS and size exclusion chromatography.