Routine Isotope-Dilution Liquid Chromatography–Tandem Mass Spectrometry Assay for Simultaneous Measurement of the 25-Hydroxy Metabolites of Vitamins D2 and D3

Background: Measurement of 25-hydroxyvitamin D2 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 chromatography–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 μL) were denatured with methanol–propanol 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 ionization MS/MS in multiple-reaction monitoring mode. Results: 25-OH D2 and D3 and the IS nearly coeluted, whereas 1α-hydroxyvitamin D3 was separated; total run time was 8 min. The interassay CVs for 25-OH D2 were 9.5% and 8.4% at 52 and 76 nmol/L, respectively, and for 25-OH D3 were 5.1% and 5.6% at 55 and 87 nmol/L, respectively. The detection limit of the present method was <4 nmol/L for both metabolites. Method comparison with a commercial RIA measuring total 25-hydroxyvitamin D showed good correlation: y = 0.97x − 2.7 nmol/L (r = 0.91). The analytical system can assay 100 samples in 12.5 h. Conclusions: This simple robust interference-free LC-MS/MS assay is suitable for routine measurement of the 25-hydroxy metabolites of vitamins D2 and D3 in human serum. The assay has been in use for 9 months and has been used to assay more than 6000 routine samples.