Measurement of vitamin D metabolites by mass spectrometry, an analytical challenge

Liquid-chromatography-tandem-mass-spectrometry (LC-MS/MS) is consideredthe gold standard for the measurement of vitamin D metabolites as it does notsuffer from most of the limitations inherent to immunoassays. In addition,LC-MS/MS offers the advantage measuring several metabolites simultaneously.Despite many advantages, LC-MS/MS methods are not free from analyticalchallenges. Aspects such as sample type, protein precipitation, analyteextraction, derivatization, chromatographic separation ionization andcapabilities of the mass spectrometer have to be considered carefully.Calibration, standardization and the use of internal standards are otherimportant issues that affect the accuracy of results. Only well designedmethods that are continuously controlled by internal and external qualitycontrol allow an accurate and stable measurement 25-hydroxy vitamin D (25(OH)D).Other metabolites, such as 1,25(OH)₂D and 24,25(OH)₂D,are not yet standardized and comparable results between laboratories aredifficult to obtain. While an extensive number of original studies and reviewarticles has addressed the performance of 25(OH)D immunoassays, the analyticalchallenges regarding the measurement of vitamin D metabolites by LC-MS/MS arenot widely known. Therefore, the aim of this review is to provide acomprehensive overview about existing LC-MS/MS methods for the measurement ofvitamin D metabolites and their analytical performance.