Analytical performance specifications for the measurement uncertainty of 24,25-dihydroxyvitamin D examinations

Etienne Cavalier (Lead / Corresponding author), Callum G. Fraser, Harjit Pal Bhattoa, Annemieke C. Heijboer, Konstantinos Makris, Samuel Vasikaran, Loreen Huyghebaert, Stéphanie Peeters, Caroline Le Goff, Markus Herrmann, Anna Carobene, IFCC-IOF Committee for Bone Metabolism

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Objectives: The exploration of the metabolites in the degradation pathways of vitamin D (VTD) has gained importance in recent years and simultaneous quantitation of twenty-five-hydroxy vitamin D (25(OH)D) mass concentration together with 24,25-dihydroxyvitamin D (24,25(OH)2D) has been proposed as a newer approach to define VTD deficiency. Yet, no data are available on 24,25(OH)2D biological variation (BV). In this study, we evaluated 24,25(OH)2D's BV on the European Biological Variation Study (EuBIVAS) cohort samples to determine if analytical performance specifications (APS) for 24,25(OH)2D could be generated. Methods: Six European laboratories recruited 91 healthy participants. 25(OH)D and 24,25(OH)2D concentrations in K3-EDTA plasma were examined weekly for up to 10 weeks in duplicate with a validated LC-MS/MS method. The Vitamin D Metabolite Ratio (24,25(OH)2D divided by 25(OH)D × 100) was also calculated at each time point. Results: Linear regression of the mean 24,25(OH)2D concentrations at each blood collection showed participants were not in steady state. Variations of 24,25(OH)2D over time were significantly positively associated with the slopes of 25(OH)D concentrations over time and the concentration of 25(OH)D of the participant at inclusion, and negatively associated with body mass index (BMI), but not with age, gender, or location of the participant. The variation of the 24,25(OH)2D concentration in participants over a 10 weeks period was 34.6%. Methods that would detect a significant change linked to the natural production of 24,25(OH)2D over this period at p<0.05 would need a relative measurement uncertainty (u%)<14.9% while at p<0.01, relative measurement uncertainty should be <10.5%. Conclusions: We have defined for the first time APS for 24,25(OH)2D examinations. According to the growing interest in this metabolite, several laboratories and manufacturers might aim to develop specific methods for its determination. The results presented in this paper are thus necessary prerequisites for the validation of such methods.

Original languageEnglish
Pages (from-to)1561–1566
Number of pages6
JournalClinical Chemistry and Laboratory Medicine
Issue number9
Early online date31 Mar 2023
Publication statusPublished - 2023


  • 24,25(OH)2D
  • 25(OH)-vitamin D
  • analytical performance specifications biological variation
  • measurement uncertainty
  • vitamin D
  • Vitamin D Metabolite Ratio (VMR)


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