A High-Throughput Platform for the Rapid Screening of Vitamin D Status by Direct Infusion-Tandem Mass Spectrometry.

Vitamin D is an important fat-soluble prohormone with pleiotropic effects on human health, such as immunomodulation of the innate and adaptive immune system. There is an unmet clinical need for a rapid screening platform for 25-hydroxyvitamin D (25OH-D) determination without chromatographic separation that offers better precision and accuracy than immunoassays. Here, we introduce a high-throughput method for assessing vitamin D status from blood specimens based on direct infusion-tandem mass spectrometry (DI-MS/MS) following click derivatization using 2-nitrosopyridine. We developed an optimized liquid-phase extraction protocol to minimize ion suppression when directly infusing serum or plasma extracts via a capillary electrophoresis system for quantitative determination of 25OH-D. Acceptable reproducibility (mean CV = 10.9%, n=412), recovery (mean = 102% at 15, 30, and 45 nmol/L), and linearity (R2 > 0.998) were achieved for 25OH-D with lower detection limits (LOD ∼ 1.2 nmol/L, S/N ∼ 3), greater throughput (∼ 3 min/sample), and less bias than a commercial chemiluminescence immunoassay prone to batch effects. There was mutual agreement in 25OH-D concentrations from reference blood samples measured by DI-MS/MS as compared to LC-MS/MS (mean bias = 7.8%, n=18). We also demonstrate that this method could reduce immunoassay misclassification of vitamin D deficiency in a cohort of critically ill children (n=30). In conclusion, DI-MS/MS offers a viable alternative to LC-MS/MS for assessment of vitamin D status in support of large-scale studies in nutritional epidemiology, as well as clinical trials to rapidly screen individual patients who may benefit from vitamin D supplementation.