Development of a method for multiple vitamin D metabolite measurements by liquid chromatography coupled with tandem mass spectrometry in dried blood spots

Abstract

There are two forms of vitamin D which are essential to the human body, i.e. vitamin D₂ (ergocalciferol) and vitamin D₃ (cholecalciferol). The inactive metabolites of vitamin D are commonly used for quantitative analysis because of their longer half-life, stability, and relatively high blood concentrations. This paper presents the development of a high-throughput and sensitive method for determining four vitamin D metabolites in dried blood spots using liquid chromatography coupled with tandem mass spectrometry. This method allows for the determination of 25(OH)D₂ and 25(OH)D₃ concentrations, as well as the epimeric form 3-epi-25(OH)D₃ and 24,25(OH)₂D₃. The analyzed material is capillary blood taken from the fingertip, deposited on filter paper. Four different chromatographic columns were tested to separate all compounds, in particular, the epimeric form. The column of choice was F5 (Phenomenex, Torrance, CA, USA). In order to prove the consistency between the results for DBS, used as an alternative biological matrix, and serum, comparative studies of these two materials were carried out in nearly 100 individuals. The results indicated their positive correlation. The evaluation of short-term stability of metabolites in DBS within the month showed no change in metabolite concentration. During the validation, the impact of the matrix on the ionization of the tested compounds was evaluated. Capillary blood and venous blood collected for different anticoagulants were also compared. The smallest differences in the results were obtained for citrate. In order to achieve a limit of quantitation of 0.2 ng ml⁻¹, sample preparation involved derivatization using a Cookson-type reagent, 4-(4′-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD).