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Furthermore, substantial progress has been made in the analysis of vitamin D metabolites and related biomarkers, such as vitamin D binding protein. Liquid chromatography tandem mass spectrometric (LC\u2013MS\/MS) methods are capable of accurately measuring multiple vitamin D metabolites in parallel. Nevertheless, only 25(OH)D and the biologically active form 1,25(OH)2D are routinely measured in clinical practice. While 25(OH)D remains the analyte of choice for the diagnosis of vitamin D deficiency, 1,25(OH)2D is only recommended in a few conditions with a dysregulated D metabolism. 24,25(OH)2D, free and bioavailable 25(OH)D, and the vitamin D metabolite ratio (VMR) have shown promising results, but technical pitfalls in their quantification, limited clinical data and the lack of reference values, impede their use in clinical practice. LC\u2013MS\/MS is the preferred method for the measurement of all vitamin D related analytes as it offers high sensitivity and specificity. In particular, 25(OH)D and 24,25(OH)2D can accurately be measured with this technology. When interpreted together, they seem to provide a functional measure of vitamin D metabolism beyond the analysis of 25(OH)D alone. The determination of VDBP, free and bioavailable 25(OH)D is compromised by unresolved analytical issues, lacking reference intervals and insufficient clinical data. Therefore, future research activities should focus on analytical standardization and exploration of their clinical value. This review provides an overview on established and new vitamin D related biomarkers including their pathophysiological role, preanalytical and analytical aspects, expected values, indications and influencing conditions.<\/jats:p>","DOI":"10.1007\/s00223-022-00961-5","type":"journal-article","created":{"date-parts":[[2022,3,3]],"date-time":"2022-03-03T11:07:26Z","timestamp":1646305646000},"page":"158-177","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":132,"title":["Vitamin D Metabolites: Analytical Challenges and Clinical Relevance"],"prefix":"10.1007","volume":"112","author":[{"given":"N.","family":"Alonso","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"S.","family":"Zelzer","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"G.","family":"Eibinger","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"M.","family":"Herrmann","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2022,3,3]]},"reference":[{"key":"961_CR1","doi-asserted-by":"publisher","first-page":"153","DOI":"10.1007\/S11154-017-9424-1","volume":"18","author":"M Holick","year":"2017","unstructured":"Holick M (2017) The vitamin D deficiency pandemic: approaches for diagnosis, treatment and prevention. 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