Improved High-Pressure Liquid Chromatographic Assay of Serum 25-Hydroxycholecalciferol and 25-Hydroxyergocalciferol After Reverse-Phase Sep-Pak C18Cartridge Preparation of Sample

A simplified method is described for extracting and purifying 25-hydroxycholecalciferol and 25-hydroxyergocalciferol from serum for quantitation by high-pressure liquid chromatography. The method involves extracting and purifying these metabolites from serum (1–10 ml) with a reverse-phase octadecylsilane bonded silica cartridge (Sep-Pak C18). This method is faster than a previously described method involving extraction with dichloromethane and purification by Sephadex LH-20 chromatography. The correlation between the two methods was excellent (r2 = 0.96, p≤0.0001). The coefficient of variation for the new method is 4.3%. The new method allows measurement of 25-hydroxyergocalciferol from human serum since both 25-hydroxycholecalciferol and 25-hydroxyergocalciferol are extracted equally. This allows the use of [3H] 25-hydroxycholecalciferol to monitor the recovery of both the D2 and the D3 forms of the metabolite.