Serum materials from three individual donors were used for 25(OH)D3 studies (6.3 ng/g, 15.3 ng/g, and 27.0 ng/g 25(OH)D3 for sera 1, 2, and 3, respectively), while a serum material from a single donor was used for 25(OH)D2 studies [0.9 ng/g 25(OH)D2]. Samples were prepared in three different sets (each on a different day), each set consisting of triplicate aliquots from each of the serum materials. Each serum material was accurately weighed (approximately 2 g) into a 50 mL glass centrifuge tube. Each sample was then spiked with appropriate weighed amounts of 25(OH)D3-d3 and 25(OH)D2-d3 to get an approximately 1:1 mass ratio of analyte to internal standard. After equilibration at room temperature for 1 h, the pH of each sample was adjusted to pH 9.8 ± 0.2 with approximately 400 μL of 0.1 g/mL carbonate buffer, pH 9.8 (approximately 200 μL of buffer per mL of liquid). The 25(OH)D3 and 25(OH)D2 were simultaneously extracted from the serum matrix with 8 mL of hexane-ethyl acetate (50:50, volume fraction). Each sample was shaken vigorously for 10 min using a mechanical shaker to allow complete mixing. The upper hexane-ethyl acetate layer was transferred to another 50 mL centrifuge tube. Hexane-ethyl acetate extraction was repeated once more with another 8 mL of solvent by shaking for 3 min. The combined extracts were dried under nitrogen at 45 °C and the residue was reconstituted with 120 μL of methanol for LC-MS/MS analysis.
Preliminary experiments were conducted to evaluate the 25(OH)D concentrations of serum materials. An initial experiment was performed where arbitrary amounts of the internal standard were chosen, and a wider range of mass ratios (0.2 to 3.0) for the standards was used. Once the approximate 25(OH)D concentration was determined, the quantity of internal standard was calculated to obtain a 1:1 mass ratio.