Assay of vitamins D2 and D3, and 25-hydroxyvitamins D2 and D3 in human plasma by high-performance liquid chromatography.

I describe a new assay that is capable of measuring vitamin D2, vitamin D3, 25-hydroxyvitamin D2, and 25-hydroxyvitamin D3 in 2 ml of plasma or serum. Plasma is extracted by the Bligh and Dyer technique [Can. J. Biochem. Physiol. 37, 911 (1959)], the lipid component is fractionated by two high-performance liquid-chromatographic systems based upon adsorption and reversed-phase chromatography, and each of the four vitamin D metabolites is measured by its absorbance at 254 nm. The method has a sensitivity limit of 0.5 mug/liter of plasma. The identity of metabolite peaks was confirmed by mass spectrometry, ultraviolet absorption spectrophotometry, and rechromatography, and there was good correlation (r=0.84) between plasma 25-hydroxyvitamin D as measured by the present method and by a protein binding assay developed in our laboratory. Mean concentrations of vitamin D and 25-hydroxyvitamin D in normal adults (n=25) in December were 2.2 +/- 1.1 (SD) and 16 +/- 3.9 (SD) mug/liter, respectively. 25-Hyroxyvitamin D2 made up 31% of the total 25-hydroxyvitamin D. Patients receiving pharmacological doses of vitamin D had values for vitamin D and 25-hydroxyvitamin D that were 10- to 100-fold normal. This method provides a rapid, reliable physico-chemical assay that appears to have advantages over existing protein binding assays and can be used to measure circulating vitamin D.