Determination of vitamin D metabolites in various biological samples through an improved chemical derivatization assisted liquid chromatography-tandem mass spectrometry approach

Abstract

Vitamin D (VD) metabolites are involved in a variety of important metabolic processes and physiological effects in organisms. Profiling of VD metabolites favors a deep understanding of the physiological role of VD. However, VD metabolites are difficult to detect due to their high chemical structural rigidity, structural similarity, and low sensitivities under liquid chromatography-tandem mass spectrometry (LC-MS). Herein, we present a chemical derivatization assisted LC-MS/MS strategy for the detection of VDs, in which 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) is employed to derivatize the conjugated diene of VD metabolites and provides sensitizing reporters for MS detection. After PTAD derivatization, the sensitivities of seven VD metabolites increased by 24–276 folds, with the limits of detection ranging from 3 to 20 pg mL⁻¹. Using this method, we achieved a sensitive and accurate quantification of 7 VD metabolites (vitamin D₂, vitamin D₃, 25-hydroxyvitamin D₂, 25-hydroxyvitamin D₃, 1,25-dihydroxyvitamin D₂, 1,25-dihydroxyvitamin D₃, and 1,24,25-trihydroxyvitamin D₃) of the VD metabolic pathway in different trace biological samples, including human serum, mouse tissues (namely liver, kidney, lung, and spleen), and cells. We believe that the present method can provide a promising tool for an in-depth analysis of VD metabolism.