Analytical methods for 25-hydroxyvitamin D: advantages and limitations of the existing assays

Vitamin D plays an important role in the absorption of calcium and phosphorus, bone metabolism. Various analytical methods are being used to measure the circulating vitamin D levels in the blood. Among all vitamin D metabolites, 25 hydroxyvitamin (25(OH) D) is the most well-known member of the vitamin D family. It is available in two forms, viz. 25 hydroxyvitamin D₃ (25(OH)D₃) and 25 hydroxyvitamin D₂(25(OH)D₂). These are stable and available in ample amounts in serum and hence are the most commonly measured during estimation of vitamin D. Although, immunoassay was the most common analytical technique, it could not differentiate between 25(OH) D₂ and 25(OH) D₃. HPLC is another low-cost technique but lacks sufficient sensitivity for determining trace amounts of 25(OH) D₂ and D₃. Further, more advanced techniques like liquid chromatography-mass spectrometry have been developed to provide better specificity, higher degree of separation and identification of different forms of vitamin D. However, this technique is not very user friendly. Thus, standardization of these analytical methods in clinical and research laboratories is essential for developing and implementing evidence based clinical guidelines. This will also enable achievement of comparable results via different methods, based on diverse principles. In this manuscript, we have presented a comprehensive overview of the efforts for standardization in vitamin D assessment, along with latest advances in methods used for 25(OH) D₂ and 25(OH) D₃ analysis. Finally, a summary of clinical trials associated with vitamin D has been presented to discuss all the analytical methods employed for measuring 25(OH) D₂ and D₃.