Determination of vitamin D₃ conjugated metabolites: a complementary view on hydroxylated metabolites

Experts typically define vitamin D deficiency levels by the determination of a circulating 25-hydroxyvitamin D₃-calcifediol prohormone. A large part of the population is characterized by deficient vitamin D levels (calcifediol < 20 ng mL^-1) despite individuals not being affected by any disorder. Cholecalciferol (vitamin D₃) and/or calcifediol supplementation is a common practice for vitamin D-deficient individuals as recommended by international scientific societies and official agencies. In the last few years, several studies have reported the presence of conjugated vitamin D₃ metabolites, mainly glucuronidation and sulfation derivatives, although simultaneous quantitative measurements involving phase I and II vitamin D metabolites have not been carried out. A quantitative method based on tandem mass spectrometry detection is proposed here for the combined determination of phase I and phase II vitamin D₃ metabolites in human serum. As phase I and phase II metabolites are preferentially ionized in different modes, a switching polarity mode was adopted to determine both groups of compounds in serum at high sensitivity levels (pg mL^-1). The validation of this proposal was successfully accomplished by following the Center for Drug Evaluation and Research (CDER) guidelines. Its applicability was tested in a cohort of volunteers with mostly deficient baseline levels. Considering the sulfated form of calcifediol, the sum of its concentrations showed sufficient baseline vitamin D levels in all individuals, suggesting that this could be a novel strategy for vitamin D deficiency definition. Therefore, phase II metabolites are proposed to be included when evaluating the vitamin D status since they provide more information about the overall status of the vitamin D endocrine system. Nevertheless, further studies are required to confirm the biological activity of these conjugated metabolites and the suitability of this strategy for the description of vitamin D deficiency.