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Vitamin D: 25OH calciferol profil (VD3 + D2) in LCMSMS

Pictogramme horloge Isabelle PETIT Pictogramme horloge January 2017

Vitamin D is a group of compounds derived from the cyclic structure of cholesterol, the most important of which are 25OHD3 or cholecalciferol and 25OHD2 or ergocalciferol.

  • 25OHD3 or cholecalciferol is primarily of endogenous origin and is formed in the skin as a result of exposure to the sun’s UV rays acting on dehydrocholesterol, and to a lesser extent of exogenous origin, derived from food products of animal origin (fish and dairy products).
  • 25OHD2 or ergocalciferol, which is of vegetable origin, is solely exogenous and is obtained from dietary fruit and vegetables.

Knowledge of the physiology of vitamin D has expanded greatly in recent years, revealing its ubiquitous role in the body that extends far beyond the phosphocalcic and bone functions: vitamin D is implicated in anti-infective, inflammation inhibitory, tumour inhibitory and cardiovascular protection processes.

To complement its existing extensive range of expert biological services, the Eurofins Biomnis laboratory has now introduced a vitamin D assay using the HPLC-MS separation technique (high-performance liquid chromatography-tandem mass spectrometry), which offers a very high analytical quality:

  • A high degree of sensitivity: the quantification threshold is 3 to 4 times lower than that of the EIA techniques.
  • A high degree of specificity:
    • For separate assays of 25OHD2 and 25OHD3
      This is extremely useful for analyses performed in relation to vitamin supplement treatments or follow-ups, especially given the wide variety of forms in which vitamin D (D2 or D3) is marketed and the great individual differences in the metabolization of these compounds. The HPLC-MS technique makes it possible to specifically assay the 25OHD3 and 25OHD2 forms, which alone will result in the 1-25OHD form, the only biologically active substance, factoring out the numerous existing inactive precursor metabolites. This assay is suitable for elucidation of a complete nutritional balance and for assessing the efficacy of a vitamin D treatment.
    • By means of an assay of the C3 epimer of 25OHD2 and of the C3 epimer of 25OHD3
      This information is invaluable in the paediatric context: the C3 epimer of 25OHD2 or of 25OHD3 may be present in more than 60% of neonates and infants of less than 1 month of age, due to enzymatic immaturity. This immaturity is mainly hepatic, involving the P450 cytochrome family (especially CYP24, CYP27A1, CYP 27B1). Studies have shown that epimerization also affects 1-25OHD. However, the action of the C3 epimer of 1-25OHD is less effective on calcium metabolism and bone mineralization.

Using the HPLC-MS technique, 25OHD3 and 25OHD2 can be isolated and quantified; these epimers are separated, assayed and, when they reach a significant concentration, are indicated in the report. This information can be invaluable to the clinician in understanding, for example, the reasons for the inefficacy of vitamin D supplements.

Eurofins Biomnis, with this benchmark technique, is providing clinicians with a relevant and informative marker in the examination of vitamin D metabolism.


  • C3 Epimers can account for a significant proportion of total circulating 25-Hydroxyvitamin D in infants, Ravinder J. Singh, The Journal of Clinical Endocrinology & Metabolism 91(8):3055–3061.
  • Fast separation of 25-hydroxyvitamine d3 from its epimer in human serum by liquid chromatography-tandem mass spectrometry showing variable C3-epimer prevalence in infants and adults, J.M.W. van den Ouweland, Ned Tijdschr Klin Chem Labgeneesk 2012; 37: 227-228
  • Evaluation, treatment, and prevention of vitamin D deficiency: an endocrine society clinical practice guideline, Michael F; Holick The Journal of Clinical Endocrinology & Metabolism June 2011
  • Measurement of circulating 25-hydroxyvitamin D: A historical review C. Le Goff, E. Cavalier, J.C. Souberbielle, Practical laboratory medicine 2 (2015) 1-14
  • Determination of vitamin D and its metabolites C.J.    Farrell Best Practice & Research Clinical Endocrinology & Metabolism ( 2013) 1-14

Article reviewed in August 2017

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