25-Hydroxyvitamin D: Analysis and clinical application

25-Hydroxyvitamin D (25-OHD) is one of the most popular tests requested by clinicians nowadays because in addition to bone diseases, many non-skeletal disorders have been suggested to be linked to vitamin D deficiency or insufficiency. Methodologies used in clinical laboratories include competitive vitamin D protein binding assays (CPBA), immunoassays, high performance liquid chromatography (HPLC), and liquid chromatography–tandem mass spectrometry (LC–MS/MS). In this review article, we introduce the basic metabolism and physiology of vitamin D, key issues in the methods for 25-OHD measurement currently used in most clinical laboratories, and clinical applications of 25-OHD testing. We conclude that although the methodologies for 25-OHD testing have improved significantly, considerable bias between different methods and laboratories still exists. Therefore, standardization of the method is critical. The optimal 25-OHD levels should be determined based on the standardized method. Also, more studies are needed to further determine the relationship between vitamin D deficiency or insufficiency and non-skeletal diseases as well as daily vitamin D dose requirement for reducing the risk of non-skeletal diseases. •25-Hydroxyvitamin D (25-OHD) can be used to assess vitamin D status.•Besides bone disorders, vitamin D may be associated with a range of other diseases.•Multiple methods have been developed for 25-OHD analysis, but bias still exists.•To reduce the bias between methods, 25-OHD assays must be standardized.•LC–MS/MS is a more accurate method for 25-OHD, but a special analyzer is needed.