Abstract
This tutorial review focuses on analytical challenges encountered with the liquid chromatography-tandem mass spectrometry determination of 25-hydroxyvitamin D, which is currently still considered the metabolite that is most representative of vitamin D status. It describes how multiple binding states of circulating 25-hydroxyvitamin D (phase II metabolites, epimers, free/bioavailable/protein-bound species) can influence the accuracy of the analytical determination. It also summarizes important chemical species that can inadvertently contribute to vitamin D status and thus cause systematic errors. These interfering endogenous and exogenous compounds might be isomers of vitamin D, constitutional isomers or isobars and the article outlines techniques to eliminate or minimize these interferences, including chromatographic separations, ion mobility spectrometry, and high-resolution mass spectrometry.