A high-throughput platform for the rapid screening of vitamin D status by direct infusion-MS/MS

J Lipid Res. 2022 May;63(5):100204. doi: 10.1016/j.jlr.2022.100204. Epub 2022 Mar 23.


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-MS/MS (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 coefficient of variation = 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 (limit of detection ∼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 with 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.

Keywords: MS; critical care; direct infusion-MS/MS; infectious disease; interlaboratory performance; method validation; nutrition; proficiency testing; vitamin D; vitamin D deficiency.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Calcifediol
  • Child
  • Chromatography, Liquid / methods
  • Humans
  • Immunoassay / methods
  • Reproducibility of Results
  • Tandem Mass Spectrometry* / methods
  • Vitamin D*
  • Vitamins


  • Vitamins
  • Vitamin D
  • Calcifediol

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