Existing standard methods for vitamin D analysis involve saponification, liquid-liquid extraction (LLE), sample clean-up, and liquid chromatography (LC)-UV determination. The most challenging aspect in vitamin D analysis is the diverse interferences from sample matrix. A large number of lipid-like compounds are co-extracted with the vitamin D, and even after extensive sample clean-up, there are still numerous interferences that co-elute and interfere with the vitamin D quantitation.
Recently, to simplify the sample preparation and to improve the analysis, a derivatization reaction with 4-Phenyl-1,2,4-triazoline-3,5-dione (PTAD) and mass spectrometry (MS) were adopted in a new AOAC standard method. This new method has provided much better analytical performance for vitamin D analysis. However, previtamin D is not measured in this new standard.
It is known that Vitamin D can thermally isomerize to Previtamin D. This transformation is reversible, and both forms are biologically active. It has been reported that the relative content of previtamin D could be up to 22% of the total vitamin D at 80oC. Therefore, it is prudent to individually determine previtamin D and vitamin D contents in the analysis of vitamin D in foods.
This application note demonstrates the determination of total vitamin D by individually measuring the vitamin D and previtamin D in food products.