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The Measurement of 3-Epimer 25-Hydroxyvitamin D by Mass Spectrometry in Clinical Specimens Detects Inconsequential Levels in Adult Subjects
  1. Mildred M. Goldman, BS, CLS,
  2. Khanh V. Viec, BS,
  3. Michael P. Caulfield, PhD,
  4. Richard E. Reitz, MD,
  5. Michael J. McPhaul, MD,
  6. Nigel J. Clarke, PhD
  1. From the Quest Diagnostics Nichols Institute, San Juan Capistrano, CA.
  1. Received October 17, 2013, and in revised form January 9, 2014.
  2. Accepted for publication January 14, 2014.
  3. Reprints: Nigel J. Clarke, PhD, Quest Diagnostics Nichols Institute, 33608 Ortega Highway, San Juan Capistrano, CA 92675. E-mail: nigel.j.clarke{at}
  4. Supplemental digital content is available for this article. Direct URL citation appears in the printed text and is provided in the HTML and PDF versions of this article on the journal’s Web site (


Background Vitamin D is derived from dietary sources or from the action of ultraviolet light on 7-dehydrocholesterol and undergoes a number of enzymatic modifications that lead to the synthesis of active vitamin D metabolites or metabolites with reduced biological activity. Among these, epimerization at the 3-hydroxyl group leads to the synthesis of 3-epimer 25-hydroxyvitamin D (3EVD). Described first in biological system experiments using in vitro incubation of vitamin D in cell culture, this molecule has been reported as having distinct activities when compared with 25-hydroxy vitamin D (25OHVD). Measurements of vitamin D have been conducted using a variety of methodologies and have led to conflicting assessments of the quantities of 3EVD3 that are measured.

Method The present article describes the development and use of a simple liquid chromatography–tandem mass spectrometry method validated by the Clinical Laboratory Improvement Amendments to quantitate 3EVD3 in 3528 subjects, including 309 children (162 are <2 years) and 232 pregnant women.

Results Our findings demonstrate that, although 3EVD3 constitutes a significant proportion of measureable 25OHVD3 in subjects younger than 1 year, 3EVD3 levels are negligible in most subjects older than 1 year.

Conclusions It is important to choose the correct 25OHVD assay dependent on the age of the patient. Patients younger than 1 year should be run on a liquid chromatography–tandem mass spectrometry assay proven to not have potential contributions from any 3EVD present in the sample.

Key Words
  • 25-hydroxy vitamin D
  • mass spectrometry
  • 3-epimer
  • pediatric
  • quantitative assay
  • adult
  • CLIA-validated assay

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