Determining vitamin D status: a comparison between commercially available assays

PLoS One. 2010 Jul 13;5(7):e11555. doi: 10.1371/journal.pone.0011555.


Background: Vitamin D is not only important for bone health but can also affect the development of several non-bone diseases. The definition of vitamin D insufficiency by serum levels of 25-hydroxyvitamin D depends on the clinical outcome but might also be a consequence of analytical methods used for the definition. Although numerous 25-hydroxyvitamin D assays are available, their comparability is uncertain. We therefore aim to investigate the precision, accuracy and clinical consequences of differences in performance between three common commercially available assays.

Methodology/principal findings: Serum 25-hydroxyvitamin D levels from 204 twins from the Swedish Twin Registry were determined with high-pressure liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (HPLC-APCI-MS), a radioimmunoassay (RIA) and a chemiluminescent immunoassay (CLIA). High inter-assay disagreement was found. Mean 25-hydroxyvitamin D levels were highest for the HPLC-APCI-MS technique (85 nmol/L, 95% CI 81-89), intermediate for RIA (70 nmol/L, 95% CI 66-74) and lowest with CLIA (60 nmol/L, 95% CI 56-64). Using a 50-nmol/L cut-off, 8% of the subjects were insufficient using HPLC-APCI-MS, 22% with RIA and 43% by CLIA. Because of the heritable component of 25-hydroxyvitamin D status, the accuracy of each method could indirectly be assessed by comparison of within-twin pair correlations. The strongest correlation was found for HPLC-APCI-MS (r = 0.7), intermediate for RIA (r = 0.5) and lowest for CLIA (r = 0.4). Regression analyses between the methods revealed a non-uniform variance (p<0.0001) depending on level of 25-hydroxyvitamin D.

Conclusions/significance: There are substantial inter-assay differences in performance. The most valid method was HPLC-APCI-MS. Calibration between 25-hydroxyvitamin D assays is intricate.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Aged
  • Aged, 80 and over
  • Biological Assay / methods*
  • Chromatography, High Pressure Liquid
  • Female
  • Humans
  • Immunoassay
  • Luminescent Measurements
  • Male
  • Mass Spectrometry
  • Middle Aged
  • Radioimmunoassay
  • Reproducibility of Results
  • Vitamin D / analogs & derivatives*
  • Vitamin D / blood


  • Vitamin D
  • 25-hydroxyvitamin D