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Mass spectrometry: A precision approach for vitamin D testing

Contributing lab leadersEtienne CavalierMarkus Herrmann

Vitamin D plays a role in multiple functions in the human body involving almost every organ system. A critical role it plays is the regulation of calcium homeostasis and bone metabolism, and low levels of vitamin D are associated with a higher risk of certain debilitating diseases.1,2

Affecting over one billion people worldwide, vitamin D deficiency presents a major global health problem and the demand for diagnostic testing has significantly increased in the last decade.3,4 In clinical practice, 25(OH)D is the most widely measured metabolite of vitamin D as it is believed to reflect vitamin D stores that are available for metabolism.5-12

Traditionally immunoassays have been the main method for testing, but due to its high sensitivity and specificity for measuring vitamin D metabolites, liquid chromatography with tandem mass spectrometry (LC-MS/MS) provides several benefits compared to other techniques, and is now viewed as the reference and preferred method. 

Article highlights:

  • Vitamin D plays a critical role in calcium homeostasis and bone metabolism and high and low levels have also been associated with a variety of debilitating diseases.
  • Vitamin D metabolite testing using mass spectrometry can provide important diagnostic and therapeutic information for clinicians, and more accurate measurements in special patient and population groups.
  • Due to its high sensitivity, precision, and reliability in detecting vitamin D metabolites, mass spectrometry has become the reference method for vitamin D testing.

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Vitamin D metabolite testing

As the most abundant vitamin D metabolite in serum, 25(OH)D is the main metabolite used for measuring vitamin D deficiency. 25(OH)D includes 25(OH)D3 (from vitamin D3), which typically accounts for 95% of circulating 25(OH)D levels, and 25(OH)D2 (from vitamin D2) which accounts for a small fraction of 25(OH)D.5 While 25(OH)D is the preferred metabolite for measuring vitamin D deficiency, additional vitamin D metabolites play a role in other metabolic and inherited disorders. 

  • 1,25(OH)2D: In some instances, the measurement of the 1,25(OH)2D metabolite is useful, including diagnosis of conditions like sarcoidosis or of acquired and inherited disorders of vitamin D and phosphate metabolism.6,7 It is also useful in the identification of CYP24A1 gene mutations which can lead to reduced CYP24A1 activity. This can be identified through elevated serum 1,25(OH)2D concentrations and decreased 24,25(OH)2D levels .8

  • 24,25(OH)2D: As it is the principal catabolite formed by CYP24A1, measurement of 24,25(OH)2D is useful for identifying patients with mutations in this gene as a cause of hypercalcemia (including idiopathic infantile hypercalcemia) and recurrent kidney stones.9 This is an important factor as in these cases vitamin D supplementation could be dangerous.10

  • Vitamin D metabolite ratio (VMR): Another biomarker that has been proposed to identify CYP24A1 activity and vitamin D catabolism, is the VMR, calculated as follows: (24,25[OH]2 D/25[OH]D)×100.11 As this ratio is not influenced by the vitamin D binding protein VDBP) concentration, the VMR demonstrates stronger association with clinical outcomes such as bone mineral density, fracture risk and all-cause mortality than 25(OH)D.12-16

The benefits of mass spectrometry for vitamin D metabolite measurement

While immunoassays are widely available and accessible with automated processes, and relatively high throughput, given the range of metabolites to consider in vitamin D testing there are several advantages to using mass spectrometry.17

Measurement and differentiation of vitamin D metabolites

Mass spectrometry is able to differentiate and quantify all clinically relevant metabolites of vitamin D, including the separation of D2 and D3 metabolites.18 It is therefore able to offer improved analytical performance over immunoassays and provide a more personalized assessment of vitamin D status.

This is particularly important for individuals at risk of vitamin D deficiency requiring monitoring and supplementation, e.g. due to matrix alteration, such as in pregnancy, patients with CKD, osteoporosis, in the ICU, or in individuals with a polymorphic variant of VDBP.19

Measurement of 25(OH)D and 24,25(OH)2D by mass spectrometry also overcomes the analytical issue of immunoassays which are unable to measure levels of 24,25(OH)2D and supports broader use of the VMR.12

Lack of cross-reactivity

A limitation of immunoassays is the interference from cross-reactive metabolites that can lead to incorrect 25(OH)D measurements.17,19 As they often have a higher affinity for 25(OH)D3, immunoassays may fail to detect 25(OH)D2 due lack of to cross-reactivity.5,20

Immunoassays can under- or over-recover total 25(OH)D2 leading to an inaccurate assessment of the vitamin D status.21 As mass spectrometry separates metabolites during the measurement process, the issue of cross-reactivity does not arise.

Epimers

Epimers are thought to exist for all major metabolites of vitamin D3 and have a near-identical molecular structure. For example, The C-3 epimer of 25(OH)D3 contributes greatly (8.7–61.1%), to circulating 25(OH)D3 levels in newborns and infants, and is a source of analytical variance when certain commercially available immunoassays are used to measure total 25(OH)D levels.22-24

The detection of C3-epi-25(OH)D3 may alter the clinical definition of vitamin D status in certain groups of individuals, which could lead to overestimation and potentially under-treatment. A well-designed mass spectrometry method allows the accurate detection of 25(OH)D, even in the presence of C-3 epimers.25

 

The future of mass spectrometry for vitamin D testing

Despite the clear advantages of mass spectrometry being used in vitamin D metabolite testing, a historical lack of standardization and use of internal standards, which may affect the accuracy of results, has been a challenge. To address this and help standardize 25(OH)D measurement, the Vitamin D External Quality Assessment Scheme (DEQAS) has been developed as an external control and contributed with others (NIST, CDC, University of Ghent) to the Vitamin D Standardization Program (VDSP). This has helped to reduce inter-laboratory variability, and greatly improved the reliability of measurements.19,26-32

With one billion people affected, vitamin D deficiency remains a global challenge with increasing demands for testing. The advantages of mass spectrometry for vitamin D metabolite measurement offer labs an opportunity to provide exact and accurate information for differential diagnosis and highly personalized patient management. Along with improved standardization reliability over the last decade, mass spectrometry can now be considered the preferred method (“gold standard”) for vitamin D testing.17,19,33,34

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  1. Hossein-nezhad & Holick. (2013). Mayo Clin Proc 88, 720-55. Paper available from https://www.mayoclinicproceedings.org/article/S0025-6196(13)00404-7/fulltext [Accessed July 2024]
  2. Grant et al. (2016). Dermatoendocrinol 16, e1187349. Paper available from https://www.tandfonline.com/doi/full/10.1080/19381980.2016.1187349 [Accessed July 2024]
  3. Cleveland Clinic. Information available from https://my.clevelandclinic.org/health/diseases/15050-vitamin-d-vitamin-d-deficiency [Accessed July 2024]
  4. Fraser et al. (2020). Calcified Tissue International 106, 3-13. Paper available from https://link.springer.com/article/10.1007/s00223-019-00620-2 [Accessed August 2024] 
  5. Farrell et al. (2012) Clinical Chemistry, 58, 531-542 Paper available from https://academic.oup.com/clinchem/article/58/3/531/5620577 [Accessed August 2024] 
  6. Holick MF et al. (2011). J Clin Endocrinol Metab 96, 1911- 1930. Paper available from https://academic.oup.com/jcem/article/96/7/1911/2833671?login=false [Accessed August 2024] 
  7. Kumar et al. (2012). Arch Biochem Biophys 523, 77-86. Paper available from https://doi.org/10.1016/j.abb.2012.03.003 [Accessed July 2024]
  8. O’Keeffe DT et al. (2016). Osteoporos Int 27, 3121-3125. Paper available from https://pubmed.ncbi.nlm.nih.gov/27129455/ [Accessed August 2024] 
  9. Dirks et al. (2018). Nutrients 10, 482. Paper available from https://www.mdpi.com/2072-6643/10/4/482 [Accessed July 2024]
  10. Kaufman et al. (2021). J Bone Miner Res 36, 1340-1350. Paper available from https://onlinelibrary.wiley.com/doi/10.1002/jbmr.4306 [Accessed July 2024]
  11. Wagner D et al. (2011) J Steroid Biochem Mol Biol 126, 72-77. Paper available from https://www.sciencedirect.com/science/article/abs/pii/S0960076011001075 [Accessed August 2024] 
  12. Herrmann M et al. (2023). Clinical Chemistry 69: 1307- 1316. https://pubmed.ncbi.nlm.nih.gov/37798100/ [Accessed August 2024] 
  13. Dugar A et al. (2023). Clinical Chemistry 69, 718-723. Paper available from https://pubmed.ncbi.nlm.nih.gov/37220642/ [Accessed August 2024] 
  14. Ginsberg C et al. (2021). Clin Chem 67, 385-393. Paper available from https://pubmed.ncbi.nlm.nih.gov/33188595/ [Accessed August 2024] 
  15. Ginsberg C et al. (2021). J Bone Miner Res 36, 2343-2350. Paper available from https://pubmed.ncbi.nlm.nih.gov/34423858/ [Accessed August 2024] 
  16. Ginsberg C et al. (2018). Bone 107, 124-130. Paper available from https://pubmed.ncbi.nlm.nih.gov/29155243/ [Accessed August 2024] 
  17. Gailor et al. (2018). Bone Rep 8, 268-273. Paper available from https://doi.org/10.1016/j.bonr.2018.05.003 [Accessed July 2024]
  18. Ihara H et al. (2018). Ann Clin Biochem 55, 647-656. Paper available from https://pubmed.ncbi.nlm.nih.gov/29534611/ [Accessed August 2024]
  19. Alonso et al. (2023). Calcif Tissue Int 112, 158-177. Paper available from https://link.springer.com/article/10.1007/s00223-022-00961-5 [Accessed July 2024]
  20. Avci E et al. (2020) Journal of medical biochemistry 39, 100-107. Paper available from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7282243/ [Accessed August 2024]
  21. Carter et al. (2016). J Steroid Biochem Mol Biol 164, 134-138. Paper available from https://doi.org/10.1016/j.jsbmb.2015.12.018 [Accessed July 2024]
  22. Lensmeyer G et al. (2012) The Journal of Clinical Endocrinology & Metabolism 97, 163-168. Paper available from https://academic.oup.com/jcem/article/97/1/163/2833297 [Accessed August 2024] 
  23. Wiebe D and Binkley N. (2014) J Clin Endocrinol Metab  99, 1117-1121. Paper available from https://pubmed.ncbi.nlm.nih.gov/24476080/ [Accessed August 2024] 
  24. Singh J. (2006) Clin Endocrinol Metab 91, 3055-3061. Paper available from https://academic.oup.com/jcem/article/91/8/3055/2656587 [Accessed August 2024] 
  25. Goldman MM et al. (2014). Journal of Investigative Medicine 62, 690-695. Paper available from https://pubmed.ncbi.nlm.nih.gov/24583901/ [Accessed August 2024] 
  26. Zelzer S et al. (2018). Journal of Laboratory and Precision Medicine 3. Paper available from https://jlpm.amegroups.org/article/view/4620/html [Accessed August 2024] 
  27. Wise SA et al. (2021). J Steroid Biochem Mol Biol 212, 105917. Paper available from https://pubmed.ncbi.nlm.nih.gov/34010687/  [Accessed August 2024] 
  28. Rabenberg M et al. (2018). BMC Public Health 18, 845. Paper available from https://bmcpublichealth.biomedcentral.com/articles/10.1186/s12889-018-5769-y [Accessed August 2024] 
  29. Tai SSC et al. (2010) Analytical Chemistry 82, 1942-1948. Paper available from https://pubmed.ncbi.nlm.nih.gov/20136128/ [Accessed August 2024] 
  30. Binkley N et al. (2017). The Journal of Steroid Biochemistry and Molecular Biology 173, 117-121. Paper available from https://pubmed.ncbi.nlm.nih.gov/27979577/ [Accessed August 2024] 
  31. DEQAS. Information available from https://www.deqas.org/ [Accessed February 2024]
  32. Briggs et al. (2023) The Journal of Steroid Biochemistry and Molecular Biology 231, 106328. Paper available from https://www.sciencedirect.com/science/article/abs/pii/S0960076023000833 [Accessed August 2024] 
  33. Volmer et al. (2015). Mass Spectrom Rev 34, 2-23. Paper available from https://doi.org/10.1002/mas.21408. [Accessed August 2024]
  34. Cavalier E et al. Clin Chem Lab Med 2019;58:197–201. Paper available from DOI: 10.1515/cclm-2019-0174 [Accessed August 2024]