Interlaboratory comparison of 25-hydroxyvitamin D assays: Vitamin D Standardization Program (VDSP) Intercomparison Study 2 - Part 1 liquid chromatography - tandem mass spectrometry (LC-MS/MS) assays - impact of 3-epi-25-hydroxyvitamin D3 on assay performance

Stephen A Wise; Johanna E Camara; Carolyn Q Burdette; Grace Hahm; Federica Nalin; Adam J Kuszak; Joyce Merkel; Ramón A Durazo-Arvizu; Emma L Williams; Andrew N Hoofnagle; Fiona Ivison; Ralf Fischer; Jody M W van den Ouweland; Chung S Ho; Emmett W K Law; Jean-Nicolas Simard; Renaud Gonthier; Brett Holmquist; Sarah Meadows; Lorna Cox; Kimberly Robyak; Michael H Creer; Robert Fitzgerald; Michael W Clarke; Norma Breen; Pierre Lukas; Étienne Cavalier; Christopher T Sempos

doi:10.1007/s00216-021-03576-1

Interlaboratory comparison of 25-hydroxyvitamin D assays: Vitamin D Standardization Program (VDSP) Intercomparison Study 2 - Part 1 liquid chromatography - tandem mass spectrometry (LC-MS/MS) assays - impact of 3-epi-25-hydroxyvitamin D₃ on assay performance

Anal Bioanal Chem. 2022 Jan;414(1):333-349. doi: 10.1007/s00216-021-03576-1. Epub 2021 Aug 25.

Authors

Stephen A Wise^{1

2}, Johanna E Camara³, Carolyn Q Burdette³, Grace Hahm³, Federica Nalin³, Adam J Kuszak⁴, Joyce Merkel⁴, Ramón A Durazo-Arvizu⁵, Emma L Williams⁶, Andrew N Hoofnagle⁷, Fiona Ivison⁸, Ralf Fischer⁹, Jody M W van den Ouweland¹⁰, Chung S Ho¹¹, Emmett W K Law¹¹, Jean-Nicolas Simard¹², Renaud Gonthier¹², Brett Holmquist¹³, Sarah Meadows^{14

15}, Lorna Cox^{14

15}, Kimberly Robyak¹⁶, Michael H Creer¹⁶, Robert Fitzgerald¹⁷, Michael W Clarke¹⁸, Norma Breen¹⁹, Pierre Lukas²⁰, Étienne Cavalier²⁰, Christopher T Sempos^{4

21}

Affiliations

¹ Office of Dietary Supplements (ODS), National Institutes of Health (NIH), Bethesda, MD, 20892, USA. stephen.wise@nih.gov.
² Scientist Emeritus, National Institute of Standards and Technology (NIST), Gaithersburg, MD, 20899, USA. stephen.wise@nih.gov.
³ Chemical Sciences Division, National Institute of Standards and Technology (NIST), Gaithersburg, MD, 20899, USA.
⁴ Office of Dietary Supplements (ODS), National Institutes of Health (NIH), Bethesda, MD, 20892, USA.
⁵ Biostatistics Core, The Sabin Research Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA, 90027, USA.
⁶ Imperial Healthcare NHS Trust, London, W6 8RF, UK.
⁷ Department of Laboratory Medicine, University of Washington, Seattle, WA, 98185, USA.
⁸ Central Manchester Foundation Trust, Manchester, M13 9WL, UK.
⁹ Chromsystems Instruments & Chemicals GmbH, 82166, Gräfelfing, Germany.
¹⁰ Canisius Wilhelmina Hospital (CWZ), 6532, SZ, Nijmegen, The Netherlands.
¹¹ Biochemical Mass Spectrometry Unit, Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong, 999077, Hong Kong.
¹² Endoceutics Inc., Québec, G1P 4P5, Canada.
¹³ Endocrine Sciences, LabCorp Specialty Testing Group, Agoura Hills, CA, 91301, USA.
¹⁴ Medical Research Council (MRC) Elsie Widdowson Laboratory (closed Dec. 2018), Cambridge, UK.
¹⁵ NIHR BRC Nutritional Biomarker Laboratory, MRC Epidemiology Unit, University of Cambridge, Cambridge, CB2 0SL, UK.
¹⁶ College of Medicine, Penn State University, Hershey, PA, 17033, USA.
¹⁷ Health Clinical Laboratories, University of California at San Diego, La Jolla, CA, 92093, USA.
¹⁸ Metabolomics Australia, Centre for Microscopy, Characterisation and Analysis, The University of Western Australia, Perth, WA, 6009, Australia.
¹⁹ Waters Technologies Ireland Ltd., Wexford, Y35 D431, Ireland.
²⁰ Clinical Chemistry, University of Liège, CHU de Liège, B-4000, Liège, Belgium.
²¹ Vitamin D Standardization Program LLC, Havre de Grace, MD, 217078, USA.

PMID: 34432104
DOI: 10.1007/s00216-021-03576-1

Abstract

An interlaboratory comparison study was conducted by the Vitamin D Standardization Program (VDSP) to assess the performance of liquid chromatography - tandem mass spectrometry (LC-MS/MS) assays used for the determination of serum total 25-hydroxyvitamin D (25(OH)D), which is the sum of 25-hydroxyvitamin D₂ (25(OH)D₂) and 25-hydroxyvitamin D₃ (25(OH)D₃). A set of 50 single-donor samples was assigned target values for concentrations of 25(OH)D₂, 25(OH)D₃, 3-epi-25-hydroxyvitamin D₃ (3-epi-25(OH)D₃), and 24R,25-dihydroxyvitamin D₃ (24R,25(OH)₂D₃) using isotope dilution liquid chromatography - tandem mass spectrometry (ID LC-MS/MS). VDSP Intercomparison Study 2 Part 1 includes results from 14 laboratories using 14 custom LC-MS/MS assays. Assay performance was evaluated using mean % bias compared to the assigned target values and using linear regression analysis of the test assay mean results and the target values. Only 53% of the LC-MS/MS assays met the VDSP criterion of mean % bias ≤ |±5%|. For the LC-MS/MS assays not meeting the ≤ |±5%| criterion, four assays had mean % bias of between 12 and 21%. Based on multivariable regression analysis using the concentrations of the four individual vitamin D metabolites in the 50 single-donor samples, the performance of several LC-MS/MS assays was found to be influenced by the presence of 3-epi-25(OH)D₃. The results of this interlaboratory study represent the most comprehensive comparison of LC-MS/MS assay performance for serum total 25(OH)D and document the significant impact of the lack of separation of 3-epi-25(OH)D₃ and 25(OH)D₃ on assay performance, particularly with regard to mean % bias.

Keywords: 24R,25-Dihydroxyvitamin D3 (24R,25(OH)3D3); 25-Hydroxyvitamin D2 (25(OH)D2); 25-Hydroxyvitamin D3 (25(OH)D3); 3-epi-25-Hydroxyvitamin D3 (3-epi-25(OH)D3); Liquid chromatography – tandem mass spectrometry (LC-MS/MS); Total serum 25-hydroxyvitamin D (25(OH)D).

MeSH terms

25-Hydroxyvitamin D 2
Chromatography, Liquid / methods
Reference Standards
Tandem Mass Spectrometry* / methods
Vitamin D* / analogs & derivatives

Substances

Vitamin D
25-Hydroxyvitamin D 2
25-hydroxyvitamin D

Grants and funding

not applicable/NIH Office of the Director