Quantification of Circulating 25-hydroxyvitamin D by Liquid Chromatography-tandem Mass Spectrometry

Hypovitaminosis D is a highly prevalent condition and quantification of serum 25-hydroxyvitamin D3 is accepted to be the most useful marker for the assessment of the individual vitamin D status. Due to the increasing awareness of the prevalence and potential health consequences of hypovitaminosis D, the request numbers for 25-hydroxyvitamin D quantification are growing rapidly in many countries. Automated protein binding assays (based on the use of vitamin D-binding protein or antibodies) for the quantification of 25-hydroxyvitamin D3 are available which enable convenient high-throughput analyses in a routine setting; there is, however, substantial concern about accuracy and analytical reliability of these assays. Several LC-MS/MS methods for the quantification of 25-hydroxyvitamin D3 in serum have been described and in a growing number of clinical laboratories this technology is used routinely for vitamin D monitoring. It is justified to assume that LC-MS/MS enables more reliable analyses of 25-hydroxyvitamin D concentrations compared to protein binding assays. In particular the ability to co-quantify the naturally occurring 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 which is derived primarily from food fortification is a relevant advantage of LC-MS/MS over protein binding assays. This review describes the background of 25-hydroxyvitamin D measurement, compares published LC-MS/MS methods, discusses problems, strengths and limitations of these assays and compares the application characteristics of LC-MS/MS with those of protein binding assays and HPLC-UV.