We received a question in our ODX AMA (Ask Me Anything) feature about the different tests available for 25-OH (hydroxy) vitamin D testing:
"Regarding the different methods of measuring 25-OH (hydroxy) vitamin D (LCMS versus immunoassay), is there a benefit to ordering the LCMS?"
Here's what the ODX Research Team was able to find out:
The LCMSMS method of measuring total 25(OH) vitamin D is currently the gold standard for evaluating vitamin D status. Immunoassays can be confounded by a number of variables but still have value if LC/MS/MS is unavailable, i.e., Beckman Coulter assay.
Recognition of widespread vitamin D insufficiency has led to increased vitamin D testing and supplementation. Biochemical assessment of vitamin D status comprises testing serum levels of the most clinically relevant metabolites 25(OH)D and 1,25(OH)2D. The 24,25(OH)2D metabolite is also clinically relevant though not widely tested for at this time.
The 25(OH)D metabolite reflects vitamin D reserves and is the most commonly tested form. The most common methodologies for measuring serum 25(OH)D are immunoassay and liquid chromatography tandem mass spectrometry (LC/MS/MS).
The immunoassay method is the most common of the two, although it may not be the most accurate. This older method can over- or under-estimate 25(OH)D or can be vulnerable to interfering factors. The LC/MS/MS method overcomes these variables and is considered the gold standard for 25(OH)D testing.
This method is considered more sensitive and specific and is the preferred method for verifying vitamin D insufficiency. Unfortunately, this method is more time-consuming and has a slower turnaround time than immunoassay, which may factor into clinical decision-making. No matter which method is utilized for assessing vitamin D status, serum levels should be monitored, and supplementation adjusted as needed to avoid deficiency or toxicity. Serum 25(OH)D is expected to increase by 0.5-1 ng/mL (1.25-2.5 nmol/L) for every 100 IU of supplementation. It is important to identify both vitamin D deficiency and toxicity as they both can have adverse consequences. Vitamin D deficiency can lead to rickets, osteomalacia, bone pain, and muscle weakness, while frank toxicity can cause hypercalcemia, hyperphosphatemia, decreased PTH levels, abdominal pain, nausea, vomiting, fatigue, weakness, and deposition of calcium in the kidney (Galior 2018). Vitamin D3 is most commonly used and may be the most efficacious for increasing serum 25(OH)D (Tripkovic 2012).
If LC/MS/MS testing is not available, there is research suggesting that immunoassay measurements of 25(OH)D have shown acceptable agreement with LC/MS/MS, as demonstrated in an evaluation of 100 random blood samples (Kocak 2015). For whichever method is available, it is always prudent to utilize the same lab when monitoring levels over time to minimize potential interfering factors. It should also be noted that the LC/MS/MS method reports D2 and D3 metabolites of 25(OH)D separately as well as reporting the total 25(OH), whereas the immunoassay reports total 25(OH) D2 and D3 together without differentiation (Quest Diagnostics).
The U.S. Vitamin D Standardization Program (VSDP) has been established to help standardize 25(OH)D testing and address variabilities in testing methodology. A comparison of 42 single-donor samples found that high levels of the D2 metabolite of 25(OH) seemed to confound results in the majority of immunoassays. Only 2 immunoassays (i.e., the Beckman Coulter and Fujirebio Inc.) and the LC/MS/MS method, were minimally affected by higher levels of vitamin D2 (Wise 2021).
Galior, Kornelia et al. “10 years of 25-hydroxyvitamin-D testing by LC-MS/MS-trends in vitamin-D deficiency and sufficiency.” Bone reports vol. 8 268-273. 23 May. 2018, doi:10.1016/j.bonr.2018.05.003
Kocak, Fatma Emel et al. “A comparison between two different automated total 25-hydroxyvitamin D immunoassay methods using liquid chromatography-tandem mass spectrometry.” Biochemia medica vol. 25,3 430-8. 15 Oct. 2015, doi:10.11613/BM.2015.044
Quest Diagnostics. Two vitamin D [25-hydroxyvitamin D or 25(OH)D] testing methods from Quest Diagnostics. https://www.questdiagnostics.com/healthcare-professionals/about-our-tests/endocrine-disorders/vitamin-d
Tripkovic, Laura et al. “Comparison of vitamin D2 and vitamin D3 supplementation in raising serum 25-hydroxyvitamin D status: a systematic review and meta-analysis.” The American journal of clinical nutrition vol. 95,6 (2012): 1357-64. doi:10.3945/ajcn.111.031070
Wise, Stephen A et al. “Vitamin D Standardization Program (VDSP) intralaboratory study for the assessment of 25-hydroxyvitamin D assay variability and bias.” The Journal of steroid biochemistry and molecular biology vol. 212 (2021): 105917. doi:10.1016/j.jsbmb.2021.105917