Mass spectrometry: A precision approach for vitamin D testing

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. 

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

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

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

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 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