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

Abstract

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 µg/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.

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