Assessment of endogenous 25-hydroxyvitamin D serum concentrations by liquid chromatography-tandem mass spectrometry in various animal species.

BACKGROUND: Mass spectrometry (MS) has become the preferential method for the analysis of vitamin D in the clinic, yet no single platform is utilized for preclinical species in drug development studies. For vitamin D, the MS platform can provide certain benefits such as applicability of a single assay for multiple species, low cost, and high specificity.

OBJECTIVES: A quantitative liquid chromatography-tandem MS (LC-MS/MS) assay for 25-hydroxyvitamin D3 (25OHD3 ) and D2 (25OHD2 ) was validated for rat, dog, mouse, and monkey, and suitability for drug development studies was assessed.

METHODS: Standards were used to determine intra- and inter-assay accuracy and precision for LC-MS/MS. Extraction recovery and carryover due to instrumentation were determined. Repeat analyses of pooled serum samples from rat, dog, mouse, and monkey were assessed for precision, and other serum samples were used to determine the normal range in each species and detect biologically relevant changes.

RESULTS: For both 25OHD3 and 25OHD2 , inaccuracy was ≤ 6%, and imprecision was ≤ 13%. Extraction recovery was 75% for 25OHD3 and 72% for 25OHD2 , and carryover was ≤ 0.1%. Measurable concentrations of 25OHD3 were recorded in serum samples from all species tested, but no 25OHD2 as diets were only fortified with 25OHD3 . This dataset provides preliminary information for the determination of RIs for 25OHD3 in rat, dog, mouse, and monkey with the LC-MS/MS platform.

CONCLUSIONS: The LC-MS/MS assay was accurate and precise for determination of endogenous concentrations of 25OHD3 in serum samples from drug development studies in rat, dog, mouse, and monkey.