Measurements of 25-hydroxyvitamin D concentrations in archived dried blood spots are reliable and accurately reflect those in plasma

J Clin Endocrinol Metab. 2014 Sep;99(9):3319-24. doi: 10.1210/jc.2014-1269. Epub 2014 Jun 2.


Context: Recognition that vitamin D might be associated with many chronic diseases has led to large-scale epidemiological and clinical studies. Dried blood spots (DBS) are a useful resource for these studies. Consequently, accurate, efficient, and inexpensive assays to quantify 25-hydroxyvitamin D (25OHD) in DBS are required.

Objective: This study evaluated the validity and reliability of a liquid chromatography-tandem mass spectrometry assay for measuring 25OHD in archived DBS and compared measurements of 25OHD in DBS with those in plasma.

Design and participants: Sixty-two participants in the Melbourne Collaborative Cohort Study who had plasma and matching DBS stored since study entry in the early 1990s were randomly selected for a study calibrating 25OHD concentrations in DBS with plasma. As part of a study of vitamin D and mortality, cancer, and diabetes, we also assessed the reliability of measurements from DBS using 500 replicates placed randomly within 31 batches run over 15 months.

Outcome measure: 25OHD concentrations were measured by liquid chromatography-tandem mass spectrometry.

Results: There was good agreement between measurements of 25OHD from DBS and plasma; R(2) = 0.73 from a regression of plasma concentration on DBS concentration. The within-batch and between-batch intraclass correlations from the 500 replicate measurements were 0.82 (95% confidence interval, 0.80, 0.85) and 0.73 (95% confidence interval, 0.68, 0.78), respectively.

Conclusions: Measuring 25OHD in DBS is a valid and reliable alternative to measuring 25OHD in sera or plasma. A simple calibration model was developed to convert measurements from DBS to equivalent plasma measurements, thus enabling comparisons against clinical reference ranges and with studies using sera or plasma samples.

Publication types

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

MeSH terms

  • Adult
  • Aged
  • Calibration
  • Chromatography, Liquid / methods
  • Chromatography, Liquid / standards
  • Diabetes Mellitus / blood
  • Diabetes Mellitus / epidemiology
  • Dried Blood Spot Testing / methods*
  • Dried Blood Spot Testing / standards*
  • Female
  • Humans
  • Male
  • Middle Aged
  • Models, Biological
  • Neoplasms / blood
  • Neoplasms / epidemiology
  • Predictive Value of Tests
  • Prospective Studies
  • Reference Values
  • Reproducibility of Results
  • Risk Factors
  • Tandem Mass Spectrometry / methods
  • Tandem Mass Spectrometry / standards
  • Vitamin D / analogs & derivatives*
  • Vitamin D / analysis
  • Vitamin D / blood


  • Vitamin D
  • 25-hydroxyvitamin D