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In addition to playing a vital role in bone health, vitamin D has been associated with many other health conditions, such as diabetes, cardiovascular diseases, cancer, multiple sclerosis, and immune system functions.
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Several epidemiologic studies have shown that the prevalence of vitamin D deficiency is widespread in the general population.
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Although, 1,25-dihydroxyvitamin D is the active form of vitamin D, 25-hydroxyvitamin D is the best marker of vitamin D status.
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Immunoassays and HPLC-MS/MS are
25-Hydroxyvitamin D Testing: Immunoassays Versus Tandem Mass Spectrometry
Section snippets
Key points
25-Hydroxyvitamin D reference intervals
Currently, 25(OH)D is considered the best indicator of the vitamin D status because contrarily to 1,25(OH)2D, its level is not dependent on parathyroid harmone (PTH) concentration and it shows low intraindividual variability because of its longer half-life of approximately 3 weeks. Unlike other vitamins, population-based vitamin D reference ranges cannot be established. This is because several factors including season of the year, gender, ethnicity, sunlight exposure, diet, age, gender, body
Harmonization of 25-hydroxyvitamin D assays
Because medical decisions regarding vitamin D deficiency are based on the specific values of 25(OH)D, it is important that different methods for the measurement of 25(OH)D are comparable and harmonized. Various studies have shown that accurate measurement of 25(OH)D is challenging and there are clinically significant differences among various 25(OH)D assays. Many factors including the inconsistent release of 25(OH)D from VDBP and its similarity with many other endogenous compounds have
25-hydroxyvitamin D assays
There are two major categories of 25(OH)D assays: nonchromatographic methods involving 25(OH)D binding protein or antibody, and chromatographic methods.12 Before 1990, only a limited number of laboratories performed 25(OH)D assays. These laboratories used in-house CPBA or HPLC with UV detection. CPBA with and without chromatographic separation of 25(OH)D were developed. These assays were laborious and many suffered from inaccuracy, poor sensitivity, imprecision, and unequal cross-reactivity
Automated immunoassays and protein-binding assays for 25-hydroxyvitamin D
Several Food and Drug Administration (FDA) approved automated immunoassays and protein-binding assays are available for the determination of 25(OH)D. These assays are available on major chemistry platforms and provide rapid turnaround time. Unlike chromatographic methods, automated assays are simple to use, often require low sample volume, and do not need a large initial capital cost and specialized expertise. Most of these assays are competitive immunoassays or protein-binding assays based on
Chromatographic assays for 25-hydroxyvitamin D
Chromatographic methods have been widely used for the assay of 25(OH)D and other metabolites of vitamin D. These methods offer several advantages, the most obvious being the separation and simultaneous measurement of several clinically relevant vitamin D metabolites, such as 25(OH)D3, 25(OH)D2, 1,25(OH)2D3, 1,25(OH)2D2, 24,25(OH)2D3, 24,25(OH)2D2, and 3-epi-25(OH)D. HPLC with UV detection, first described in 1978, was an earlier chromatographic method for the assay of vitamin D metabolites.
Comparison of immunoassays and protein-binding assays with chromatographic methods
Several studies have compared 25(OH)D immunoassays and protein-binding assays with chromatographic methods involving UV detection or mass spectrometry.15, 28, 29, 30, 31, 32 Until recently, immunoassays and protein-binding assays suffered from significant inaccuracies and imprecision. Several factors, including inconsistent release of 25(OH)D from VDBP, uneven cross-reactivity of antibody with 25(OH)D3 and 25(OH)D2, interferences from endogenous compounds, and lack of standard materials
Summary
Vitamin D deficiency has been associated with poor bone health and a myriad of other illnesses including diabetes, cardiovascular diseases, autoimmune disorders, and cancer. Because of these associations and the important role of vitamin D in health and disease, clinical laboratories have seen tremendous growth in 25(OH)D testing in recent years. To classify a patient into a right category of deficiency or sufficiency, accurate measurement of 25(OH)D is of the utmost importance. Although
References (37)
- et al.
Hydroxyvitamin D assays: an historical perspective from DEQAS
J Steroid Biochem Mol Biol
(2018) - et al.
25-Hydroxyvitamin D assays: potential interference from other circulating vitamin D metabolites
J Steroid Biochem Mol Biol
(2016) - et al.
Measurement of circulating 25-hydroxyvitamin D: a historical review
Pract Lab Med
(2015) - et al.
Vitamin D metabolite profiling using liquid chromatography-tandem mass spectrometry (LC-MS/MS)
J Steroid Biochem Mol Biol
(2016) - et al.
A rapid method for the separation of vitamin D and its metabolites by ultra-high performance supercritical fluid chromatography-mass spectrometry
J Chromatogr A
(2016) - et al.
Measurement of 25-OH-vitamin D in human serum using liquid chromatography tandem-mass spectrometry with comparison to radioimmunoassay and automated immunoassay
J Chromatogr B Analyt Technol Biomed Life Sci
(2010) - et al.
Analytical measurement and clinical relevance of vitamin D(3) C3-epimer
Clin Biochem
(2013) - et al.
Variability in the analysis of 25-hydroxyvitamin D by liquid chromatography-tandem mass spectrometry: the devil is in the detail
Clin Chim Acta
(2012) Vitamin D deficiency
N Engl J Med
(2007)The vitamin D deficiency pandemic: approaches for diagnosis, treatment and prevention
Rev Endocr Metab Disord
(2017)
Screening for vitamin D deficiency: a systematic review for the U.S. Preventive Services Task Force
Ann Intern Med
Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline
J Clin Endocrinol Metab
The 2011 report on dietary reference intakes for calcium and vitamin D from the Institute of Medicine: what clinicians need to know
J Clin Endocrinol Metab
Serum 25(OH)-vitamin D level in children: is there a need to change the reference range based on 2011 Institute of Medicine report?
Clin Pediatr (Phila)
The 2011 report on dietary reference intakes for calcium and vitamin D
Public Health Nutr
Practical guidelines for the supplementation of vitamin D and the treatment of deficits in Central Europe: recommended vitamin D intakes in the general population and groups at risk of vitamin D deficiency
Endokrynol Pol
Assessment criteria for vitamin D deficiency/insufficiency in Japan: proposal by an expert panel supported by Research Program of Intractable Diseases, Ministry of Health, Labour and Welfare, Japan, The Japanese Society for Bone and Mineral Research and The Japan Endocrine Society [Opinion]
Endocr J
Determination of vitamin D status by radioimmunoassay with an 125I-labeled tracer
Clin Chem
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2020, Revue NeurologiqueCitation Excerpt :Methods involving immunoassays, protein-binding assays, high-performance liquid chromatography (HPLC)-UV, and liquid chromatography – tandem mass spectrometry (LC-MS/MS) are available for the measurement of 25(OH)D [52]. Nevertheless, the gold standard for the measurement of vitamin D and of its metabolites is HPLC and especially LC-MS/MS [52,53]. Other methods deployed (non-chromatographic) usually lack accuracy and underestimate 25(OH)D levels [54–56], but, in view of financial and technical considerations automated immunoassays are often utilized [53].
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2020, Annales d'EndocrinologieCitation Excerpt :However, establishing reference values depends closely on the standardisation of dosages or even discrimination between different forms of vitamin D that do not necessarily have the same biological effects. Different automated immunoassays of vitamin D do not distinguish between cholecalciferol and ergocalciferol (for review [24]). The LCMS assay can therefore serve as a reference method for separating these 2 molecules but it remains difficult to separate their epimers especially 3-épi-25(OH)D3 [25].
Development and application of a LC–MS/MS assay for simultaneous analysis of 25-hydroxyvitamin-D and 3-epi-25-hydroxyvitamin-D metabolites in canine serum
2020, Journal of Steroid Biochemistry and Molecular BiologyCitation Excerpt :3-epi-1α,25(OH)2D can occur via C-3 epimerization of 1α,25(OH)2D, but also via 1α-hydroxylation of 3-epi-25(OH)D, in the same manner that conversion of 25(OH)D to 1α,25(OH)2D occurs [26–28]. Liquid chromatography tandem mass spectrometry (LC–MS/MS) is the gold-standard approach to quantify vitamin D metabolites [24,29]. LC–MS/MS offers superior specificity and can distinguish between vitamin D2 and D3 metabolites, unlike immunoassays.
Disclosure: The author has nothing to disclose.