Simplified HPLC method for quantitation of vitamin D in blood serum.

A single-step high performance liquid chromatography (HPLC) method for vitamin D quantitation in blood serum is described. Methanol/hexane extraction, silica Sep-Pak purification, differential dissolution, filtration, and the use of a precolumn in place of an injector sample loop allow for using only one (reversed phase) HPLC step instead of the usual two. Detection limit with the HPLC system used was about 1 micrograms/L of serum. Recovery of vitamin D3 added to serum ranged from 90% to 109%. Intra and interassay coefficient of variation values were 8.1% and 15.4%, respectively.