Revolutionizing vitamin D assessment with the power of LC-MS/MS

Mass spectrometry, particularly liquid chromatography-tandem mass spectrometry (LC-MS/MS), offers superior sensitivity, specificity, and accuracy over traditional immunoassays in assessing vitamin D levels by precisely measuring individual metabolites including 25(OH)D2, 25(OH)D3, and C-3 epimers, enabling personalized vitamin D management, especially in vulnerable populations like neonates and patients with specific health conditions, while advancements in automation and standardization are making it the emerging gold standard for clinical vitamin D testing worldwide. In this exclusive interview, SelectScience^® speaks to Dr. Benjamin Lilienfeld, Lifecycle Leader Serum Work Area Systems, Senior Vice President, Roche to learn more.

SelectScience: Can you explain the key advantages of mass spectrometry in assessing vitamin D levels compared to traditional immunoassays?

Lilienfeld: Thanks to its high sensitivity, specificity and accuracy, mass spectrometry offers several key advantages over immunoassays when it comes to assessing vitamin D levels. 25(OH)D, also known as 25-hydroxyvitamin D or calcidiol, is the major circulating metabolite of vitamin D in the body.

A limitation of immunoassays is the interference from cross-reactive metabolites that can lead to incorrect 25(OH)D measurements.^{1, 2} While immunoassays rely on antibodies that can sometimes cross-react with structurally similar compounds, mass spectrometry, particularly when coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS), provides unparalleled specificity. As mass spectrometry separates metabolites during the measurement process, the issue of cross-reactivity does not arise. This is achieved by separating molecules and then identifying them based on their unique mass-to-charge ratio, allowing for the precise measurement of individual vitamin D metabolites like 25(OH)D2 and 25(OH)D3, while effectively distinguishing them from potential interferents.³

Furthermore, LC-MS/MS enables the simultaneous quantification of multiple vitamin D metabolites in a single analysis, offering a more comprehensive understanding of a patient's vitamin D status. Although the standard mass spectrometry report provides the total 25(OH)D measurement, the system measures 25(OH)D2 and 25(OH)D3 levels. This is particularly important in regions where vitamin D2 supplementation is common. Unlike most immunoassays, which tend to have a higher affinity for 25(OH)D3 than 25(OH)D2, mass spectrometry provides consistent accuracy and avoids the misclassification of vitamin D status when both forms are present.⁴

Consequently, due to its enhanced specificity and ability to differentiate between analytes, LC-MS/MS generally demonstrates higher accuracy and lower bias, particularly at low vitamin D concentrations, which is crucial for accurate diagnosis and monitoring. Finally, the inherent specificity of mass spectrometry provides a stronger foundation for standardization across different laboratories and methods, contributing to more consistent and comparable results in vitamin D testing.

SelectScience: How does LC-MS/MS contribute to more personalized vitamin D management?

Lilienfeld: LC-MS/MS may offer improved analytical performance over immunoassays in individuals at risk of vitamin D deficiency requiring vitamin D monitoring and supplementation (e.g., due to matrix alteration, such as in pregnancy, patients with CKD (Chronic Kidney Disease), osteoporosis, in the ICU (Intensive Care Unit) or in individuals with a polymorphic variant of VDBP (Vitamin D Binding Protein)).⁵ Immunoassays have demonstrated variable analytical performance in such individuals and results were often in poor agreement with validated LC-MS/MS results.⁶ Thus, in general, LC-MS/MS offers enhanced precision and accuracy in measuring vitamin D and its metabolites. The quantification of vitamin D metabolites using LC-MS/MS also represents a significant advance in personalized patient management.⁷ Due to the variability of 25(OH)D levels between individuals, a personalized assessment of vitamin D status presents superior diagnostic specificity as opposed to relying on a fixed 25(OH)D cut-off for vitamin D deficiency.⁸ The Vitamin D Metabolite Ratio (VMR) of 24,25(OH)2D and 25(OH)D has been proposed for more personalized assessment as it is an indicator of vitamin D catabolism. Not influenced by 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. The measurement of 24,25 Vitamin D requires the advanced analytical performance of LC-MS/MS.⁹

SelectScience: How does the cobas^® Mass Spec solution account for C-3 epimers in vitamin D analysis, and why is this important for neonates and infants?

Lilienfeld: Epimers are thought to exist for all major metabolites of vitamin D3 and have a near-identical molecular structure.¹⁰ The cobas^® Mass Spec solution can account for C-3 epimers in vitamin D analysis through its high resolution liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) technology. This technique allows for the chromatographic separation of the C-3 epimers of 25(OH)D2 and 25(OH)D3 from their non-epimerized forms.¹¹ By separating these compounds based on their slightly different chemical properties during chromatography, it can specifically identify and quantify each form individually based on their unique mass-to-charge ratios. This detailed level of analysis is particularly significant when assessing vitamin D status in neonates and infants. They often exhibit considerably higher circulating concentrations of the C-3 epimer of 25(OH)D3 compared to adults, sometimes representing a substantial fraction of the total measured 25(OH)D. Traditional immunoassays which often lack the ability to distinguish between 25(OH)D and its C-3 epimer due to their structural similarities, can consequently overestimate the true levels of 25(OH)D. The accurate differentiation and quantification of C-3 epimers provided by the cobas^® Mass Spec solution offers a more precise assessment of true vitamin D status in neonates and infants, which is crucial for informed clinical management and a deeper understanding of vitamin D metabolism during this critical stage of development.

SelectScience: What kind of sample preparation is required for vitamin D measurement using the cobas^® Mass Spec solution?

Lilienfeld: The cobas^® Mass Spec solution provides end-to-end automation of the sample preparation process. After being centrifuged, as with clinical chemistry or Elecsys platforms, the biological sample (for example serum or plasma for vitamin D analysis) is loaded onto the system, and the instrument takes over. This means that all the steps necessary to prepare the sample for analysis are performed automatically, without significant manual intervention.

SelectScience: Do you see mass spectrometry becoming the gold standard for vitamin D analysis in clinical laboratories?

Lilienfeld: 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 gold standard for vitamin D testing.^{12, 13, 14, 15} Significant progress in automation and standardization is now transforming mass spectrometry from a highly specialized research tool into a more accessible technology for routine clinical and diagnostic applications.

The cobas^® Mass Spec solution incorporates automated sample handling, streamlined data acquisition, and increasingly standardized protocols, significantly reducing the manual steps and variability associated with earlier methods. This progress enables laboratories to analyze a greater number of samples with improved speed and consistency. As automated and standardized mass spectrometry-based assays become more widely adopted, the capacity to keep pace with the global demand for vitamin D testing increases substantially, facilitating more effective monitoring of population trends and the implementation of targeted interventions to combat vitamin D deficiency on a worldwide scale.¹⁶

Related reading:

Dr. Benjamin Lilienfeld discusses Roche mass spectrometry solution launch

Revolutionizing diagnostics with automated, integrated, and standardized clinical mass spectrometry

References:

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^[4] Farrell C-JL, Martin S, McWhinney B, et al. State-of-the-Art Vitamin D Assays: A Comparison of Automated Immunoassays with Liquid Chromatography–Tandem Mass Spectrometry Methods. Clinical Chemistry 2012; 58: 531-542. DOI: 10.1373/clinchem.2011.172155.

^[5] Alonso et al. (2023). Calcif Tissue Int 112, 158-177. Paper available from https://link.springer.com/article/10.1007/s00223-022-00961-5.

^[6] Farrell Christopher-John L., Martin Steven, McWhinney Brett, et al. State of the Art Vitamin D Assays: A Comparison of Automated Immunoassays with Liquid Chromatography Tandem Mass Spectrometry Methods. Clinical Chemistry 2012; 58: 531-542. DOI: 10.1373/clinchem.2011.172155.

^[7] Herrmann Markus, Zelzer Sieglinde, Cavalier Etienne, et al. Functional Assessment of Vitamin D Status by a Novel Metabolic Approach: The Low Vitamin D Profile Concept. Clinical Chemistry 2023; 69: 1307-1316. DOI: 10.1093/clinchem/hvad151.

^[8] Herrmann Markus, Zelzer Sieglinde, Cavalier Etienne, et al. Functional Assessment of Vitamin D Status by a Novel Metabolic Approach: The Low Vitamin D Profile Concept. Clinical Chemistry 2023; 69: 1307-1316. DOI: 10.1093/clinchem/hvad151.

^[9] Herrmann Markus, Zelzer Sieglinde, Cavalier Etienne, et al. Functional Assessment of Vitamin D Status by a Novel Metabolic Approach: The Low Vitamin D Profile Concept. Clinical Chemistry 2023; 69: 1307-1316. DOI: 10.1093/clinchem/hvad151.

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^[16] Volmer et al. (2015). Mass Spectrom Rev 34, 2-23. Paper available from https://doi.org/10.1002/mas.21408.