Review Article| Volume 38, ISSUE 3, P553-564, September 2018

Development of a 25-Hydroxyvitamin D Liquid Chromatography–Tandem Mass Spectrometry Assay, Cleared by the Food and Drug Administration, via the De Novo Pathway

  • Nicole V. Tolan
    SCIEX, 500 Old Connecticut Path, Framingham, MA 01701.
    Department of Anatomic and Clinical Pathology, Tufts University School of Medicine, 800 Washington Street, Boston, MA 02111, USA

    SCIEX Diagnostics, 500 Old Connecticut Path, Framingham, MA 01701, USA
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      1. Tolan NV. Vitamin D. In: Rifai N, editor. New England journal of medicine knowledge+ and American association for clinical chemistry learning lab adaptive learning program. 2017. Available at: Accessed July 12, 2017.

        • Risteli J.
        • Winter W.
        • Kleerekoper M.
        • et al.
        Burtis C.A. Ashwood E.R. Bruns D.A. Tietz textbook of clinical chemistry and molecular diagnostics vol. 5th edition. Saunders Elsevier, St. Louis (MO)2012: 1765
      2. McCudden C. Vitamin D. 2012. Available at: Accessed July 12, 2017.

      3. Ross A.C. Taylor C.L. Yaktine A.L. Del Valle B.H. Dietary Reference Intakes for Calcium and Vitamin D Institute of Medicine (US) Committee to Review Dietary Reference Intakes for Vitamin D and Calcium. National Academies Press (US), Washington (DC)2011
        • Holick M.F.
        The D-lemma: to screen or not to screen for 25-hydroxyvitamin D concentrations.
        Clin Chem. 2010; 56: 729-731
        • Thomas M.K.
        • Lloyd-Jones D.M.
        • Thadhani R.I.
        • et al.
        Hypovitaminosis D in medical inpatients.
        N Engl J Med. 1998; 338: 777-783
        • Kolata G.
        D is for Dilemma. Why are so many people popping vitamin D?.
        The New York Times, 2017
        • Holick M.F.
        • Binkley N.C.
        • Bischoff-Ferrari H.A.
        • et al.
        Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline.
        J Clin Endocrinol Metab. 2011; 96: 1911-1930
        • Scott M.G.
        • Gronowski A.M.
        • Reid I.R.
        • et al.
        Vitamin D: the more we know, the less we know.
        Clin Chem. 2015; 61: 462-465
        • Shahangian S.
        • Alspach T.D.
        • Astles J.R.
        • et al.
        Trends in laboratory test volumes for Medicare Part B reimbursements, 2000-2010.
        Arch Pathol Lab Med. 2014; 138: 189-203
        • Lensmeyer G.
        • Poquette M.
        • Wiebe D.
        • et al.
        The C-3 epimer of 25-hydroxyvitamin D(3) is present in adult serum.
        J Clin Endocrinol Metab. 2012; 97: 163-168
        • Strathmann F.G.
        • Sadilkova K.
        • Laha T.J.
        • et al.
        3-epi-25 hydroxyvitamin D concentrations are not correlated with age in a cohort of infants and adults.
        Clin Chim Acta. 2012; 413: 203-206
        • van den Ouweland J.M.
        • Beijers A.M.
        • van Daal H.
        Overestimation of 25-hydroxyvitamin D3 by increased ionisation efficiency of 3-epi-25-hydroxyvitamin D3 in LC-MS/MS methods not separating both metabolites as determined by an LC-MS/MS method for separate quantification of 25-hydroxyvitamin D3, 3-epi-25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in human serum.
        J Chromatogr B Analyt Technol Biomed Life Sci. 2014; 967: 195-202
        • Yang Y.
        • Rogers K.
        • Wardle R.
        • et al.
        High-throughput measurement of 25-hydroxyvitamin D by LC-MS/MS with separation of the C3-epimer interference for pediatric populations.
        Clin Chim Acta. 2016; 454: 102-106
        • Carter G.D.
        25-hydroxyvitamin D: a difficult analyte.
        Clin Chem. 2012; 58: 486-488
        • Shu I.
        • Pina-Oviedo S.
        • Quiroga-Garza G.
        • et al.
        Influence of vitamin D2 percentage on accuracy of 4 commercial total 25-hydroxyvitamin D assays.
        Clin Chem. 2013; 59: 1273-1275
        • Le Goff C.
        • Peeters S.
        • Crine Y.
        • et al.
        Evaluation of the cross-reactivity of 25-hydroxyvitamin D2 on seven commercial immunoassays on native samples.
        Clin Chem Lab Med. 2012; 50: 2031-2032
        • Li L.
        • Zeng Q.
        • Yuan J.
        • et al.
        Performance evaluation of two immunoassays for 25-hydroxyvitamin D.
        J Clin Biochem Nutr. 2016; 58: 186-192
      4. National Institutes of Health, Office of Dietary Supplements. Vitamin D Initiative. Available at: Accessed February, 2016.

        • Mineva E.M.
        • Schleicher R.L.
        • Chaudhary-Webb M.
        • et al.
        A candidate reference measurement procedure for quantifying serum concentrations of 25-hydroxyvitamin D(3) and 25-hydroxyvitamin D(2) using isotope-dilution liquid chromatography-tandem mass spectrometry.
        Anal Bioanal Chem. 2015; 407: 5615-5624
        • Tolan N.V.
        • Yoon E.J.
        • Brady A.R.
        • et al.
        Price of High-Throughput 25-Hydroxyvitamin D Immunoassays: Frequency of Inaccurate Results.
        Journal of Applied Lab Med. 2017; 2: 461-463
        • Annesley T.M.
        • Cooks R.G.
        • Herold D.A.
        • et al.
        Clinical mass spectrometry-achieving prominence in laboratory medicine.
        Clin Chem. 2016; 62: 1-3
        • Landau M.
        An interview with David Millington.
        Clin Chem. 2016; 62: 12-19
        • Jones G.
        • Prosser D.E.
        • Kaufmann M.
        25-hydroxyvitamin D-24-hydroxylase (CYP24A1): its important role in the degradation of vitamin D.
        Arch Biochem Biophys. 2012; 523: 9-18
      5. FDA 510(k) Submission: waters masstrak immunosuppressants kit. Available at: Accessed December, 2017.

      6. Titus K. Mass spec up front for pain management testing: Interest growing in oral fluid testing as alternative to urine testing. 2016. Available at: Accessed June, 2016.

        • Herold D.A.
        • Fitzgerald R.L.
        Immunoassays for testosterone in women: better than a guess?.
        Clin Chem. 2003; 49: 1250-1251
      7. FDA Overview of the process for clearance and approval of mass spectrometry-based in vitro diagnostic devices. Available at: Accessed January 29, 2018.

        • Lathrop J.T.
        • Jeffery D.A.
        • Shea Y.R.
        • et al.
        US Food and Drug Administration perspectives on clinical mass spectrometry.
        Clin Chem. 2016; 62: 41-47
      8. U.S. Food and Drug Administration. Public workshop on mass spectrometry in the clinic: regulatory considerations surrounding validation of liquid chromatography-mass spectrometry Based Devices. 2016. Available at: Accessed September, 2017.

        • Regnier F.E.
        • Skates S.J.
        • Mesri M.
        • et al.
        Protein-based multiplex assays: mock presubmissions to the US Food and Drug Administration.
        Clin Chem. 2010; 56: 165-171
      9. (CLSI document EP05-A3)Evaluation of precision of quantitative measurement procedures; approved guideline. 3rd edition. Clinical Laboratory Standards Institute (CLSI), Wayne (PA)2014
      10. (CLSI document EP06-A)Evaluation of the linearity of quantitative measurement procedures: a statistical approach; approved guideline. 1st edition. Clinical Laboratory Standards Institute (CLSI), Wayne (PA)2003
      11. (CLSI document EP17–A2)Evaluation of detection capability for clinical laboratory measurement procedures; approved guideline. 2nd edition. Clinical Laboratory Standards Institute (CLSI), Wayne (PA)2012
      12. (CLSI document EP07-A2)Interference testing in clinical chemistry; approved guideline. 2nd edition. Clinical Laboratory Standards Institute (CLSI), Wayne (PA)2005
      13. (CLSI document EP07–A2)Defining, establishing, and verifying reference intervals in the clinical laboratory; approved guideline. 3rd edition. Clinical Laboratory Standards Institute (CLSI), Wayne (PA)2010
      14. (CLSI document C62-A)Liquid chromatography-mass spectrometry methods; approved guidelines. Clinical Laboratory Standards Institute, Wayne (PA)2014