18] Quantitation of vitamin D2, vitamin D3, 25-hydroxyvitamin D2, and 25-hydroxyvitamin D3 in human milk

Abstract

This chapter describes the procedures which individually measures vitamins D2, vitamin D3, 25-hydroxyvitamin D3 (25-OHD2), and 25-hydroxyvitamin D3 (25-OHD3) in human milk. Two milliliters of whole milk is placed in a 50-ml screw-top glass centrifuge tube. To each milk sample is added 1,000 cpm of the following radioactive standards for monitoring the analytical recoveries of the assay: [3H]vitamin D3, 25-OH[3H]D2, and 25-OH[3H]D3. After vortex mixing of the milk samples, the lipids are extracted using potassium carbonate: ethyl acetate. Next, 0.8 milk volume of distilled water is added, mixed well, and followed by the addition of 3 milk volumes ethyl acetate with further mixing. The samples are then centrifuged at 1,000 g for 5 min at 20° followed by removal of the upper organic phase by aspiration. Three additional milk volumes of ethyl acetate are reintroduced to each sample and the procedure repeated. The organic phases are combined and dried under N2 in preparation for the alkaline backwash. Silica Sep-Pak preparative chromatography cartridges are used to eliminate some contaminating lipids and also to separate vitamin D from 25-OHD. The Sep-Pak cartridges are prepared and equilibrated by eluting 3 ml methanol, 4 ml isopropanol, and finally 7 ml hexane. The vitamin D fraction is further purified using a silica Bond Elute cartridge. The vitamin D fraction from the Bond Elute cartridge is purified further using two separate high-performance liquid chromatography (HPLC) systems. The first system is normal-phase developed in hexane: dichloromethane: isopropanol (49.25:49.25:0.5, v/v) with a flow rate of 2 ml/min. Final quantitation of all vitamin D compounds is performed using competitive protein binding analysis (CPBA). These assays are all performed in a nonequilibrium fashion as to greatly increase their sensitivity.