Sensitive Vitamin D Metabolite Measurement

Blood samples were drawn at the examination into serum separator tubes without anticoagulant, centrifuged at 1430 × g for 15 minutes, and 1.8 milliliters of serum transferred to Nunc Cryotubes and stored at −80C for future analysis. 100 μL of serum was mixed with 25D₃–[²H₆] and 24R,25-(OH)₂D₃–[²H₆] isotopic internal standards dissolved in 5% bovine serum albumin (IsoSciences, Inc., King of Prussia, PA). Total 25D₃ and 24,25D₃ were extracted away from DBP and other serum binding factors by protein precipitation with 250 μL methanol and cleared by centrifugation. Vitamin D metabolites were isolated from extracted supernatants by solid phase extraction chromatography (Strata C-18E 96-well SPE plates, Phenomenex, Inc., Torrences, CA), and eluted with 1 mL ethyl acetate containing 0.1 mg/mL 4-Phenyl-1,2,4-triazole-3,5-dione (PTAD). PTAD-derivatized samples were dried under vacuum and redissolved with 100 μL of 50% ethanol. Samples were then analyzed for vitamin D metabolites using reverse phase chromatography coupled to tandem mass spectrometry in multiple reaction monitoring mode (intra-assay CV 1.1% and 3.5% for 25D₃ and 24,25(OH)₂D₃,respectively). Assays were calibrated using 25D₃ and 24R,25-(OH)₂D₃ commercial standards (Cerilliant, Inc., Round Rock, TX). Intact PTH levels were measured using the Cobas electrochemiluminescense immunoassay on the Modular Analytics E170 automated analyzer (Roche Diagnostics, Indianapolis, IN, USA; inter-assay CV 2.5%). Additional method details are described in the online Supplementary Materials file.