Shedding Light on Advances in Vitamin D Testing

New testing technologies eliminate uncertainty and increase clinical utility of vitamin D analysis

In consideration of national Vitamin D Day - which took place on November 2nd - it’s a perfect time to shine light on the evolution of methods to track accuracy and clinical utility of this essential compound. Vitamin D testing has progressed from ligand-binding assays to advanced immunoassay techniques and LC-MS/MS methods, with new technologies aimed at clarifying testing accuracy and expanding clinical applications.

Why is vitamin D essential?

Vitamin D is essential to assist the body in accessing minerals for bone and tissue growth and maintenance. It does this by regulating blood levels of calcium, phosphorous, and magnesium, boosting their bioavailability to various locations throughout the body

How is vitamin D obtained?

There are two main sources of Vitamin D – endogenous, produced in the skin upon exposure to sunlight, and exogenous, obtained from the diet and/or supplementation. Vitamin D has slightly different chemical structures depending on the source. Vitamin D2 (ergocatciferol) comes largely from plants while Vitamin D3 (cholecatciferol) originates from animals. While the D2 form is found in fortified foods and in most vitamin D supplements, D3 is the form produced in the body and is used in select supplements. Both forms are effective for conversion by the liver and the kidneys to 1,25-dihydroxyvitamin D, the bioactive form.

How is vitamin D tested?

Traditional tests for Vitamin D involve ligand-binding assays, either radioimmunoassay or competitive protein-binding assays. There are several problems associated with these assays that cause persistent challenges in accuracy and precision.

  • The assays rely on detection in an aqueous, water-soluble environment. Vitamin D, specifically 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 are poorly soluble, and extraction methods to overcome insolubility suffer from inconsistencies and low precision.
  • These inconsistencies in have traditionally led to low precision across laboratories, producing patient results that are largely dependent on the laboratory used for analysis.
  • Ligand-binding assays cannot distinguish between the 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 forms, which has made it difficult to determine, for instance, whether Vitamin D therapies are due to non-compliance or the result of malabsorption.

Under or overestimation of Vitamin D due to sensitivity and specificity issues can have pronounced effects on patient health and treatment outcomes.

What are the latest vitamin D testing techniques?

High-performance liquid chromatography (HPLC) techniques have been developed that solve the accuracy and precision issues of previous assays. Although considered a new standard for measurement, the excess time, cost, and volume requirements of HPLC (~1 mL), have made standard-flow HPLC less than ideal for most labs.

Tandem-mass spectrometry coupled with liquid chromatography (LC-MS/MS) has emerged as a superior technique and has now graduated to “gold standard” status. The technique has supplanted previous approaches for several key reasons, including:

  • Higher accuracy and precision measurements owing to internal standards of defined concentrations.
  • Faster processing times with lower sample volume requirements.
  • Ability to resolve both 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3, helping to clarify the results of Vit D2 deficiency and replacement therapies.
  • Better inter-lab and inter-assay comparisons with other assays, providing more precise reference values.

In addition, the latest LC-MS/MS techniques are not hindered by lot-to-lot variability and reagent availability issues compared with previous assays.

New Vitamin D testing technologies

Although LC-MS/MS Vitamin D testing is not yet universally implemented throughout the clinical chemistry world, a growing number of labs now employ laboratory developed tests (LDTs) developed and validated in-house. These tests adhere to standardized metrics for calibration and performance set forth by CLIA. Inter-laboratory proficiency testing is performed to ensure consistency between testing sites.

On the commercial front, the first FDA-cleared Vitamin D assay kit designed for mass spectrometry – the Vitamin D 200M Assay Kit from SCIEX - was commercialized in 2017. This In Vitro Diagnostic (IVD) for clinical use was the first of its kind and was vetted through the CDC Vitamin D Standardization Certification Program (VDSCP). Coupled with the SCIEX Topaz mass spectrometry system and ClearCore MD software, the kit provides a ready-made, easy to run solution for clinical chemistry labs.

The MassTrak Vitamin D Solution from Waters was developed and validated to run on the Waters ACQUITY UPLC I-Class/Xevo TQD IVD system. To speed sample preparation and delivery to the UPLC, the system employs the Tecan Freedom 100/4 EVO offline automated liquid handling device.

Thermo Scientific recently launched the Cascadian SM Clinical Analyzer. This fully automated CE-marked IVD system utilizes LC-MS/MS with online sample processing and TurboFlow technology. The system has a throughput of 25 samples per hour, high-precision, and a wide linear range for detection of both Vit D2 and Vit D3.


Several recent developments are helping to lead the way to more accurate and precise Vitamin D testing. There are now NIST (National Institute of Standards and Technology) assigned Standard Reference Materials (SRMs) available for use in testing. NIST/CDC have aligned commercial calibration materials available for routine calibration and validation of testing methods as well. 

Although challenges remain in removing biases in Vitamin D testing, initiatives including the Vitamin D standardization certification program (VDSCP) are helping close the gaps in quality assurance and standardization.