Dealing with Vitamin D Assay Variability

Inconsistencies among vitamin D testing methods highlight the need for standardization across labs

Raeesa Gupte, PhD

Testing for vitamin D deficiency is on the rise. Between 2006 and 2015, a consistent increase in vitamin D laboratory tests was reported in 13 studies across seven countries.1 The rise in testing may be due to increasing awareness of the association between vitamin D deficiency and several health conditions including cardiovascular disorders, cancer, and depression. To meet the growing demand for vitamin D testing, diagnostic laboratories have developed a range of testing methods. 

Metabolism and Transport of Vitamin D

Vitamin D is a steroidal hormone with two major biological forms—vitamin D2 or ergocalciferol and Vitamin D3 or cholecalciferol. Vitamin D3 is a 27-carbon cholesterol derivative synthesized endogenously in the presence of sunlight. Vitamin D2 is an exogenously procured 28-carbon ergosterol derivative that is less bioactive than vitamin D3. D2 also has lower affinity for the vitamin D binding protein, resulting in faster clearance from blood circulation. However, both forms undergo 25-hydroxylation in the liver to form 25(OH)D2 and 25(OH)D3, respectively. The 25(OH)D compounds are the major circulating forms of vitamin D. 25-hydroxy vitamin D undergoes further hydroxylation in the kidney to form the biologically active 1,25-dihydroxy vitamin D. The enzyme epimerase can further metabolize 25-hydroxy vitamin D and 1,25-dihydroxy vitamin D into their C-3 epimers.

Under normal physiological conditions, approximately 85 percent of vitamin D metabolites are bound to the vitamin D binding protein (VDBP), 15 percent are bound to albumin, and 0.03 percent are unbound. Vitamin D metabolites, specifically the stable 25-hydroxylated forms of vitamin D, are used as biomarkers of vitamin D status.2

Types of vitamin D assays

Early assays for measuring vitamin D were based on the principle of competitive protein binding. Since then, immunoassay, chromatography, and mass spectrometry-based methods have all been used to measure vitamin D levels in blood.2 


Several automated immunoassay-based methods of vitamin D testing have been approved by the US Food and Drug Administration. These include two-step chemiluminescent immunoassays or radioimmunoassays. In the first step, 25(OH)D and other hydroxylated metabolites are extracted from serum by dissociating them from their binding proteins. In the second step, immunoassay is performed using anti-25(OH)D antibodies and a radioactive or luminescent tracer. Total 25(OH)D concentration is calculated based on a standard curve. Assays based on similar principles including chemiluminescent microparticle immunoassay, electrochemiluminescence, and enzyme immunoassay are commercially available. 


Chromatographic methods have been used to directly measure vitamin D metabolites without antibody binding. High performance liquid chromatography (HPLC) relies on precipitation, extraction, and phase separation of vitamin D metabolites using organic solvents. Chromatographic phase separation occurs due to varying affinities of metabolites to the solid phase and mobile phase. The peaks of the chromatogram are then measured by a UV detector and quantified. 

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the currently accepted gold standard for vitamin D testing. Following chromatographic separation, samples are introduced into a mass spectrometer. Samples are then fragmented, ionized, and analyzed based on their mass to charge ratio. Besides being able to quantify the various vitamin D metabolites simultaneously, LC-MS/MS can also measure free and protein-bound 25(OH)D. 

Challenges in vitamin D testing

Despite their low dynamic range, immunoassay techniques constitute 90 percent of routine vitamin D analyses. Automated immunoassay platforms are widely used across clinical diagnostic laboratories due to their speed, ease of use, and high throughput. However, discrepancies in total vitamin D concentrations have been reported between the various commercially available assays.3 These discrepancies may be due to differing antibody specificities and cross-reactivity to other vitamin D metabolites. There is high variability in the ability of commercial immunoassays to accurately measure 25(OH)D2, resulting in overestimation or underestimation of total 25(OH)D levels.4 Designing antibodies that are co-specific for 25(OH)D2 and 25(OH)D3 and have low cross-reactivity to other metabolites or steroid hormones circulating in blood has proven challenging. 

Chromatographic methods like HPLC can effectively separate 25(OH)D2, 25(OH)D3, and other vitamin D metabolites. Therefore, they may provide a more accurate insight into vitamin D status. They also have higher specificity and dynamic ranges than immunoassays. However, they tend to be cumbersome, low throughput, and require more sample and greater technical expertise than competitive binding assays. To make the tests more cost effective and less labor intensive, commercial HPLC kits were developed. Early HPLC kits only measured 25(OH)D3 levels, but newer kits based on reverse phase HPLC can measure 25(OH)D2 and 25(OH)D3. 

“There is a lack of a clear consensus on whether total 25(OH)D is an accurate indicator of vitamin D status.”

Like HPLC, LC-MS/MS also requires specialized equipment, large sample volume, extensive sample preparation, and specialized staff. In addition, dihydroxylated metabolites with low ionization efficiencies and interference from isomeric compounds may present significant challenges. Chemically converting low abundance, low ionization compounds into those with improved thermostability and ionization efficiency may resolve these issues.5 Commercial LC-MS/MS assays that measure 25(OH)D2 and 25(OH)D3 are also increasingly becoming available.6 These kits include calibration and quality control materials, internal standards, sample preparation materials, mobile phases, and analytical columns. 

Currently, vitamin D status is reported in terms of total 25(OH)D, without distinguishing between the protein-bound versus free forms of the metabolites. However, there is a lack of a clear consensus on whether total 25(OH)D is an accurate indicator of vitamin D status in health and disease.8 The fraction of unbound versus protein-bound 25(OH)D may vary as a result of liver disease, renal disease, and pregnancy. In addition, VDBP isoforms (GC1f, Gc1s, Gc2) may differ in their affinity for 25(OH)D. Therefore, measuring unbound versus protein-bound levels in addition to the total concentration of vitamin D may provide important insights into physiological and clinical conditions. Despite the availability of ELISA and LC-MS/MS assays that directly measure free 25(OH)D levels, this approach has not yet been adopted into clinical practice. 

Overcoming inconsistencies

“Laboratory tests used to measure vitamin D status must be accurate and consistent to ensure correct diagnosis and optimal treatment of patients. Test results of individuals should not vary drastically between laboratories or based on the type of assay used.”

The variability in vitamin D testing methods highlights the need for standardization. Laboratory tests used to measure vitamin D status must be accurate and consistent to ensure correct diagnosis and optimal treatment of patients. Test results of individuals should not vary drastically between laboratories or based on the type of assay used. Therefore, the Centers for Disease Control and Prevention (CDC) have established the Vitamin D Standardization Certification Program.5,7 To ensure consistency across clinical laboratories, the program provides reference measurements for total 25(OH)D and monitors the precision and performance of tests over time. The CDC issues certificates to manufacturers and clinical laboratories for each analytical system evaluated. 


 The low dynamic range and high variability of immunoassay techniques makes them less favorable for vitamin D testing. HPLC and mass spectrometry, once the favored tools of analytical chemists, are now finding increasing applications in clinical diagnostic laboratories. The availability of HPLC and LC-MS/ MS kits can make these techniques more efficient and user-friendly, thus promoting widespread acceptance across diagnostic laboratories. With the ability to directly measure free 25(OH)D levels, these techniques can provide physiologically relevant information about common clinical conditions. 


1.Rockwell, M., Kraak, V., Hulver, M. & Epling, J. Clinical management of low vitamin D: A scoping review of physicians’ practices. Nutrients 10 (2018). 

2. Arneson, W. L. & Arneson, D. L. Current methods for routine clinical laboratory testing of vitamin D levels. Lab. Med. 44, e38–e42 (2013). 

3. Lee, J. H., Choi, J.-H., Kweon, O. J. & Park, A. J. Discrepancy between Vitamin D Total Immunoassays due to Various Crossreactivities. J. Bone Metab. 22, 107 (2015). 

4. Freeman, J., Wilson, K., Spears, R., Shalhoub, V. & Sibley, P. Performance evaluation of four 25-hydroxyvitamin D assays to measure 25-hydroxyvitamin D2. Clin. Biochem. 48, 1097– 1104 (2015). 

5. Galior, K., Ketha, H., Grebe, S. & Singh, R. J. 10 years of 25-hydroxyvitamin-D testing by LC-MS/MS-trends in vitamin-D deficiency and sufficiency. Bone Reports 8, 268–273 (2018). 

6. Zelzer, S., Goessler, W. & Herrmann, M. Measurement of vitamin D metabolites by mass spectrometry, an analytical challenge. J. Lab. Precis. Med. 3, 99–99 (2018). 

7. Vitamin D Standardization-Certification Program (VDSCP) | CDC.

8. Bikle, D. D. & Schwartz, J. Vitamin D binding protein, total and free vitamin D levels in different physiological and pathophysiological conditions. Frontiers in Endocrinology 317 (2019).