Mass spectrometry assays of vitamin D metabolites

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as the gold standard technique with the capability for reliable, accurate, and high-throughput quantification of circulating vitamin D metabolites. The complexity of vitamin D metabolism has driven the continuous development of the basic steps involved in LC-MS/MS methodology, including sample extraction, derivatization, separation, and mass spectrometry, used to interrogate the role of vitamin D nutrition and metabolism in human health and disease states. From the perspective of both the high-throughput clinical chemistry laboratory as well as basic research applications, this chapter describes how LC-MS/MS can be optimized to provide sensitive and specific measurements of 25-hydroxyvitamin D, along with other low-abundance metabolites including 24,25-dihydroxyvitamin D and 1,25-dihydroxyvitamin D, simultaneously. Furthermore, we describe the advantages of using LC-MS/MS in studying mouse models of vitamin D metabolism, and how this work has contributed to an improved understanding of the pathophysiology of certain vitamin D-related disease states.