{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,8]],"date-time":"2026-07-08T16:23:13Z","timestamp":1783527793085,"version":"3.55.0"},"reference-count":25,"publisher":"Bioscientifica","issue":"3","content-domain":{"domain":["journals.bioscientifica.com"],"crossmark-restriction":true},"short-container-title":[],"published-print":{"date-parts":[[2026,4,1]]},"abstract":"<jats:p>Equilibrium dialysis followed by liquid chromatography\u2013tandem mass spectrometry is considered the gold standard for the isolation and quantification of free hormones. However, this technique has not been described for the analysis of free 25-hydroxyvitamin D3. This study investigated analytical challenges, focusing on stability and adsorption of 25-hydroxyvitamin D3 in aqueous buffer environments, to be considered prior to method development of equilibrium dialysis for free vitamin D. Experiments revealed that 25-hydroxyvitamin D3 is highly prone to adsorption, and to a minor extent degradation, resulting in severe analyte loss already upon introduction of 25-hydroxyvitamin D3 into the aqueous environment, far exceeding observations in serum where protein binding provides significant protection. While chemical degradation only slightly contributes, strong surface adsorption accounts for the majority of 25-hydroxyvitamin D3 loss observed. In contrast, the addition of methanol to the buffer environment completely mitigated analyte loss, confirming the severe adsorption of 25-hydroxyvitamin D3 to recipient surfaces. However, incorporating methanol as a buffer component fundamentally altered experimental conditions, thereby potentially disrupting the physiological equilibrium between 25-hydroxyvitamin D3, vitamin D-binding protein, and albumin, and by extension the free fraction of vitamin D. Consequently, the large extent of, primarily, adsorption of 25-hydroxyvitamin D3 in aqueous environments represents an important consideration in setting up equilibrium dialysis experiments for free vitamin D. These findings additionally highlight a critical need for novel analytical strategies that prevent adsorption while maintaining physiological equilibrium in the assessment of free vitamin D.<\/jats:p>","DOI":"10.1530\/jme-25-0207","type":"journal-article","created":{"date-parts":[[2026,4,16]],"date-time":"2026-04-16T10:15:22Z","timestamp":1776334522000},"update-policy":"https:\/\/doi.org\/10.1530\/crossmarkpolicy-6","source":"Crossref","is-referenced-by-count":0,"title":["Adsorption represents a critical challenge in the quantification of free 25-hydroxyvitamin D3"],"prefix":"10.1530","volume":"76","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7342-8724","authenticated-orcid":true,"given":"Nick","family":"Narinx","sequence":"first","affiliation":[{"name":"1"},{"name":"2"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6863-7903","authenticated-orcid":true,"given":"Tom","family":"De Waal","sequence":"additional","affiliation":[{"name":"1"},{"name":"2"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-4787-3637","authenticated-orcid":true,"given":"Jennifer Afrakoma","family":"Nyamaah","sequence":"additional","affiliation":[{"name":"1"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6050-6677","authenticated-orcid":true,"given":"Karel","family":"David","sequence":"additional","affiliation":[{"name":"1"},{"name":"3"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5159-3238","authenticated-orcid":true,"given":"Joeri","family":"Walravens","sequence":"additional","affiliation":[{"name":"4"},{"name":"5"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1398-5489","authenticated-orcid":true,"given":"Tom","family":"Fiers","sequence":"additional","affiliation":[{"name":"6"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1584-4965","authenticated-orcid":true,"given":"Bruno","family":"Lapauw","sequence":"additional","affiliation":[{"name":"4"},{"name":"5"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4153-4757","authenticated-orcid":true,"given":"Brigitte","family":"Decallonne","sequence":"additional","affiliation":[{"name":"1"},{"name":"3"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8676-7709","authenticated-orcid":true,"given":"Frank","family":"Claessens","sequence":"additional","affiliation":[{"name":"7"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4852-4340","authenticated-orcid":true,"given":"Jaak","family":"Billen","sequence":"additional","affiliation":[{"name":"2"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1395-0104","authenticated-orcid":true,"given":"Dirk","family":"Vanderschueren","sequence":"additional","affiliation":[{"name":"1"},{"name":"3"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7076-061X","authenticated-orcid":true,"given":"Pieter","family":"Vermeersch","sequence":"additional","affiliation":[{"name":"2"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1079-2860","authenticated-orcid":true,"given":"Leen","family":"Antonio","sequence":"additional","affiliation":[{"name":"1"},{"name":"3"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"416","published-online":{"date-parts":[[2026,4,15]]},"reference":[{"key":"2026070811515819700_bib1","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1007\/s11914-012-0098-z","article-title":"Vitamin D and bone","volume":"10","author":"Bikle","year":"2012","journal-title":"Curr Osteoporos Rep"},{"key":"2026070811515819700_bib2","doi-asserted-by":"crossref","first-page":"1109","DOI":"10.1210\/er.2018-00126","article-title":"Skeletal and extraskeletal actions of vitamin D: current evidence and outstanding questions","volume":"40","author":"Bouillon","year":"2019","journal-title":"Endocr Rev"},{"key":"2026070811515819700_bib3","doi-asserted-by":"crossref","DOI":"10.1016\/j.jsbmb.2019.105563","article-title":"Nutritional rickets: historic overview and plan for worldwide eradication","volume":"198","author":"Bouillon","year":"2020","journal-title":"J Steroid Biochem Mol Biol"},{"key":"2026070811515819700_bib4","doi-asserted-by":"crossref","first-page":"1168","DOI":"10.3390\/nu14061168","article-title":"Calcifediol (25OH vitamin D(3)) deficiency: a risk factor from early to old age","volume":"14","author":"Bouillon","year":"2022","journal-title":"Nutrients"},{"key":"2026070811515819700_bib5","doi-asserted-by":"crossref","first-page":"81","DOI":"10.1007\/s42000-019-00169-7","article-title":"The measurement of vitamin D metabolites: part I-metabolism of vitamin D and the measurement of 25-hydroxyvitamin D","volume":"19","author":"Makris","year":"2020","journal-title":"Hormones (Basel)"},{"key":"2026070811515819700_bib6","doi-asserted-by":"crossref","first-page":"427","DOI":"10.1053\/j.ajkd.2024.03.025","article-title":"Vitamin D metabolites before and after kidney transplantation in patients who are anephric","volume":"84","author":"Jorgensen","year":"2024","journal-title":"Am J Kidney Dis"},{"key":"2026070811515819700_bib7","doi-asserted-by":"crossref","first-page":"96","DOI":"10.1038\/s41574-021-00593-z","article-title":"The health effects of vitamin D supplementation: evidence from human studies","volume":"18","author":"Bouillon","year":"2022","journal-title":"Nat Rev Endocrinol"},{"key":"2026070811515819700_bib8","doi-asserted-by":"crossref","first-page":"232","DOI":"10.1210\/edrv-10-3-232","article-title":"The free hormone hypothesis: a physiologically based mathematical model","volume":"10","author":"Mendel","year":"1989","journal-title":"Endocr Rev"},{"key":"2026070811515819700_bib9","doi-asserted-by":"crossref","first-page":"543","DOI":"10.1007\/s00018-022-04562-1","article-title":"Role of sex hormone-binding globulin in the free hormone hypothesis and the relevance of free testosterone in androgen physiology","volume":"79","author":"Narinx","year":"2022","journal-title":"Cell Mol Life Sci"},{"key":"2026070811515819700_bib10","doi-asserted-by":"crossref","first-page":"230","DOI":"10.1097\/MED.0000000000000891","article-title":"Use of calculated free testosterone in men: advantages and limitations","volume":"31","author":"Nyamaah","year":"2024","journal-title":"Curr Opin Endocrinol Diabetes Obes"},{"key":"2026070811515819700_bib11","doi-asserted-by":"crossref","first-page":"254","DOI":"10.1093\/clinchem\/hvae202","article-title":"Measurement of serum free vitamin D concentrations: importance, challenges, and the emerging role of mass spectrometry","volume":"71","author":"Alexandridou","year":"2025","journal-title":"Clin Chem"},{"key":"2026070811515819700_bib12","doi-asserted-by":"crossref","first-page":"1631","DOI":"10.1210\/jc.2013-3874","article-title":"A comparison of measured and calculated free 25(OH) vitamin D levels in clinical populations","volume":"99","author":"Schwartz","year":"2014","journal-title":"J Clin Endocrinol Metab"},{"key":"2026070811515819700_bib13","doi-asserted-by":"crossref","first-page":"111","DOI":"10.1530\/EC-16-0078","article-title":"The variation in free 25-hydroxy vitamin D and vitamin D-binding protein with season and vitamin D status","volume":"6","author":"Olerod","year":"2017","journal-title":"Endocr Connect"},{"key":"2026070811515819700_bib14","doi-asserted-by":"crossref","DOI":"10.1016\/j.jsbmb.2022.106206","article-title":"The plasma free fraction of 25-hydroxyvitamin D(3) is not strongly associated with 25-hydroxyvitamin D(3) clearance in kidney disease patients and controls","volume":"226","author":"Best","year":"2023","journal-title":"J Steroid Biochem Mol Biol"},{"key":"2026070811515819700_bib15","doi-asserted-by":"crossref","first-page":"176","DOI":"10.1006\/abio.1998.2586","article-title":"Determination of non-protein-bound plasma 1,25-dihydroxyvitamin D by symmetric (rate) dialysis","volume":"258","author":"van Hoof","year":"1998","journal-title":"Anal Biochem"},{"key":"2026070811515819700_bib16","doi-asserted-by":"crossref","DOI":"10.1016\/j.jsbmb.2020.105672","article-title":"Distribution of free 25OHD in elderly population based on LC-MS\/MS","volume":"200","author":"Wang","year":"2020","journal-title":"J Steroid Biochem Mol Biol"},{"key":"2026070811515819700_bib17","doi-asserted-by":"crossref","DOI":"10.1042\/BSR20221326","article-title":"Development of free 25-hydroxyvitamin D3 assay method using liquid chromatography-tandem mass spectrometry","volume":"42","author":"Ishimine","year":"2022","journal-title":"Biosci Rep"},{"key":"2026070811515819700_bib18","doi-asserted-by":"crossref","first-page":"954","DOI":"10.1210\/jcem-63-4-954","article-title":"Assessment of the free fraction of 25-hydroxyvitamin D in serum and its regulation by albumin and the vitamin D-binding protein","volume":"63","author":"Bikle","year":"1986","journal-title":"J Clin Endocrinol Metab"},{"key":"2026070811515819700_bib19","doi-asserted-by":"crossref","first-page":"2167","DOI":"10.1210\/jc.2017-02360","article-title":"Reassessing free-testosterone calculation by liquid chromatography-tandem mass spectrometry direct equilibrium dialysis","volume":"103","author":"Fiers","year":"2018","journal-title":"J Clin Endocrinol Metab"},{"key":"2026070811515819700_bib20","doi-asserted-by":"crossref","first-page":"1584","DOI":"10.1373\/clinchem.2008.117366","article-title":"Preanalytical stability of 25(OH)-vitamin D3 in human blood or serum at room temperature: solid as a rock","volume":"55","author":"Wielders","year":"2009","journal-title":"Clin Chem"},{"key":"2026070811515819700_bib21","doi-asserted-by":"crossref","first-page":"773","DOI":"10.1093\/clinchem\/27.5.773","article-title":"Stability of vitamin D metabolites in human blood serum and plasma","volume":"27","author":"Lissner","year":"1981","journal-title":"Clin Chem"},{"key":"2026070811515819700_bib22","doi-asserted-by":"crossref","first-page":"1931","DOI":"10.1373\/clinchem.2008.111526","article-title":"Serum 25-OH vitamin D2 and D3 are stable under exaggerated conditions","volume":"54","author":"Lewis","year":"2008","journal-title":"Clin Chem"},{"key":"2026070811515819700_bib23","doi-asserted-by":"crossref","first-page":"321","DOI":"10.11613\/BM.2013.039","article-title":"Effect of sample type, centrifugation and storage conditions on vitamin D concentration","volume":"23","author":"Colak","year":"2013","journal-title":"Biochem Med"},{"key":"2026070811515819700_bib24","doi-asserted-by":"crossref","first-page":"910","DOI":"10.3389\/fendo.2019.00910","article-title":"Vitamin D binding protein: a historic overview","volume":"10","author":"Bouillon","year":"2020","journal-title":"Front Endocrinol"},{"key":"2026070811515819700_bib25","doi-asserted-by":"crossref","first-page":"617","DOI":"10.3390\/pharmaceutics13050617","article-title":"Comprehensive stability study of vitamin D3 in aqueous solutions and liquid commercial products","volume":"13","author":"Temova Rakusa","year":"2021","journal-title":"Pharmaceutics"}],"container-title":["Journal of Molecular Endocrinology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/jme.bioscientifica.com\/view\/journals\/jme\/76\/3\/JME-25-0207.xml","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/jme.bioscientifica.com\/downloadpdf\/journals\/jme\/76\/3\/JME-25-0207.xml","content-type":"text\/html","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/journals.bioscientifica.com\/jme\/article-pdf\/doi\/10.1530\/JME-25-0207\/507025\/jme-25-0207.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/journals.bioscientifica.com\/jme\/article-pdf\/doi\/10.1530\/JME-25-0207\/507025\/jme-25-0207.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,8]],"date-time":"2026-07-08T15:52:08Z","timestamp":1783525928000},"score":1,"resource":{"primary":{"URL":"https:\/\/journals.bioscientifica.com\/jme\/article\/76\/3\/e250207\/75657\/Adsorption-represents-a-critical-challenge-in-the"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,4,1]]},"references-count":25,"journal-issue":{"issue":"3","published-print":{"date-parts":[[2026,4,1]]}},"URL":"https:\/\/doi.org\/10.1530\/jme-25-0207","relation":{},"ISSN":["1479-6813","0952-5041"],"issn-type":[{"value":"1479-6813","type":"electronic"},{"value":"0952-5041","type":"print"}],"subject":[],"published":{"date-parts":[[2026,4,1]]},"article-number":"e250207"}}