Vitamin D, commonly known as the “sunshine vitamin”, is essential for maintaining calcium metabolism and bone health. The major physiologic function of Vitamin D is to maintain the blood calcium and phosphorus levels within the normal range in order to maintain metabolic functions that are essential for most life processes, Vitamin D3 and Vitamin D2 are metabolized to 25-OH-Vitamin D3 and 25-OH-Vitamin D2 in the liver by the 25-OHase enzyme. The quantitation of these metabolites is widely used for determining of vitamin D status in human. The Vitamin D status has a clinical significance in a variety of disorders which related to hormones. The measurements of the serum concentration of 25-OH-Vitamin D3 and 25-OH-Vitamin D2 have been used for differential diagnosis of hypocalcemic and hypercalcemic metabolic bone disorders.

The analysis of these metabolites is considered to be difficult. Some reasons of the difficulties are below.

•           The low concentrations of 25-OH Vitamin D metabolites in serum (level of μg/L).

•           The low stability in the presence of light and heat.

•           The presence of other metabolites which have chemical similarity.

•           The large amount of other related neutral lipids (such as cholesterol)