Vitamin D status assessed by a validated HPLC method: within and between variation in subjects supplemented with vitamin D3

Jette Jakobsen, Anette Bysted, Rikke Andersen, T. Bennett, Christine Brot, S. Bugel, K. D. Cashman, E. Denk, M. Harrington, B. Teucher, T. Walczyk, Lars Ovesen

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Objective. The aim of this study was to develop and validate a high-pressure liquid chromatography (HPLC) method for assessing vitamin D status as 25-hydroxyvitamin D2 (S-25OHD2) and 25-hydroxyvitamin D3 (S-25OHD3) in serum. Material and methods. We assessed the within- and between-subject variation of vitamin D status in serum samples from four different dietary intervention studies in which subjects (n=92) were supplemented with different doses of vitamin D3 (5-12 g/day) and for different durations (4-20 months). Results. The HPLC method was applicable for 4.0-200 nmol S-25OHD/L, while the within-day and between-days variations were 3.8 % and 5.7 %, respectively. There was a concentration-dependent difference between results obtained by a commercial radioimmunoassay and results from the HPLC method of -5 to 20 nmol 25OHD/L in the range 10-100 nmol 25OHD/L. The between-subject variation estimated in each of the four human intervention studies did not differ significantly (p=0.55). Hence, the pooled standard deviation was 15.3 nmol 25OHD3/L. In the studies with 6-8 samplings during 7-20 months of supplementation, the within-subject variation was 3.9-7.2 nmol 25OHD3/L, while vitamin D status was in the range 47-120 nmol/L. Conclusions. The validated HPLC method was applied in samples from human intervention studies in which subjects were supplemented with vitamin D3. The estimated standard deviation between and within subjects is useful in the forthcoming decision on setting limits for optimal vitamin D status.
Original languageEnglish
JournalScandinavian Journal of Clinical & Laboratory Investigation
Issue number2
Pages (from-to)190-197
Publication statusPublished - 2009


  • HPLC
  • vitamin D status
  • 25-hydroxyvitamin D
  • serum
  • RIA

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