title: 25-Hydroxyvitamin D assays: Potential interference from other circulating vitamin D metabolites.
abstract: The vitamin D External Quality Assessment Scheme (DEQAS) for 25-hydroxyvitamin D (25-OHD) has approximately 1100 participants in 53 countries using 26 different methods or variants of methods (October 2014). In April 2015, the scheme was extended to cover 24,25-dihydroxyvitamin D (24,25(OH)2D). Since 2013, the 25-OHD scheme has been accuracy-based with values assigned by the NIST reference measurement procedure (RMP). DEQAS is uniquely placed to assess the accuracy (bias) and specificity of 25-OHD methods in a routine laboratory setting. Other vitamin D metabolites are known to interfere in 25-OHD assays and DEQAS has distributed samples spiked with 3-epi-25-OHD3(52.4nmol/L), 24R,25(OH)2D3(14.4nmol/L) and 24S,25(OH)2D3(57.9nmol/L). The 3-epimer showed a cross reactivity of 56% in a competitive protein binding assay but was not detected in any antibody-based methods. Not all HPLC/UV or LC-MS/MS methods were able to resolve 3-epi-25-OHD3from 25-OHD3and thus overestimated total 25-OHD. The cross reactivity of 24R,25(OH)2D3(24S,25(OH)2D3) ranged from <5% (<5%) to 548% (643%) in ligand binding assays. Both 24-hydroxylated metabolites were resolved by HPLC/UV and LC-MS/MS methods and thus caused no complications in the measurement of 25-OHD. Most antibodies to 25-OHD are known to cross-react with dihydroxylated metabolites but interference in some assays was far greater than expected. This may be related to the anomalous behaviour of exogenously added metabolites in these 25-OHD methods.
authors.affiliation: Charing Cross Hospital, Fulham Palace Road, London W6 8RF, UK. Electronic address: b.carter1@which.net.
authors.forename: G D
authors.initials: GD
authors.lastname: Carter
authors.affiliation: Charing Cross Hospital, Fulham Palace Road, London W6 8RF, UK.
authors.forename: J C
authors.initials: JC
authors.lastname: Jones
authors.affiliation: Charing Cross Hospital, Fulham Palace Road, London W6 8RF, UK.
authors.forename: J
authors.initials: J
authors.lastname: Shannon
authors.affiliation: Charing Cross Hospital, Fulham Palace Road, London W6 8RF, UK.
authors.forename: E L
authors.initials: EL
authors.lastname: Williams
authors.affiliation: Department of Biomedical and Molecular Sciences, Queen's University, ON K7L 3N6, Canada.
authors.forename: G
authors.initials: G
authors.lastname: Jones
authors.affiliation: Department of Biomedical and Molecular Sciences, Queen's University, ON K7L 3N6, Canada.
authors.forename: M
authors.initials: M
authors.lastname: Kaufmann
authors.affiliation: National Institutes of Health, Bethesda, MD 20892-7, USA.
authors.forename: C
authors.initials: C
authors.lastname: Sempos
mesh_terms: D015650:24,25-Dihydroxyvitamin D 3 / Q000032:analysis / Q000097:blood / Q000378:metabolism; D002112:Calcifediol / Q000032:analysis / Q000097:blood* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D012680:Sensitivity and Specificity; D013237:Stereoisomerism; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014807:Vitamin D / Q000031:analogs & derivatives / Q000032:analysis / Q000097:blood / Q000378:metabolism
chemical_list: D014807:Vitamin D; D015650:24,25-Dihydroxyvitamin D 3; C104450:25-hydroxyvitamin D; D002112:Calcifediol
keywords: 24,25-Dihydroxyvitamin D; 25-Hydroxyvitamin D; 3-epi-25-Hydroxyvitamin D; DEQAS; Interference