Routine Isotope-Dilution Liquid Chromatography– Tandem Mass Spectrometry Assay for Simultaneous Measurement of the 25-Hydroxy

and D3 (25-OH D2 and D3) is essential for investigating vitamin D deficiency. Competitive binding techniques are unable to distinguish between the 2 metabolites and suffer from interference from other hydroxy metabolites of vitamin D. Methods: We used isotope-dilution liquid chromatog-raphy–tandem mass spectrometry (ID-LC-MS/MS) for routine determination of 25-OH D2 and D3 with a stable-isotope–labeled internal standard (IS). Serum samples (100 L) were denatured with methanol–propa-nol containing IS, vortex-mixed, extracted into hexane, and dried under nitrogen. The reconstituted extract was chromatographed on a BDS C8 HPLC column, and the metabolites and IS were detected by electrospray ion-ization MS/MS in multiple-reaction monitoring mode