Quantification of vitamin D and 25-hydroxyvitamin D in soft tissues by liquid chromatography-tandem mass spectrometry

Inadequate data on tissue distribution of vitamin D and its metabolites remains a barrier to defining health outcomes of vitamin D intake and 25-hydroxyvitamin D (25(OH)D) status. The purpose of this study was to develop a method for the analysis of vitamin D2 (ergocalciferol), vitamin D3 (cholecalciferol), 25(OH)D2, and 25(OH)D3 in soft tissues, and determine distribution in select tissues from a dose-response study of vitamin D2 and vitamin D3 in rats. Liver, gastrocnemius muscle, and epididymal fat homogenates were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionization following liquid-liquid extraction, solid-phase extraction, and derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD). A dose-response was observed in most tissues for vitamin D and 25(OH)D from both vitamers. Vitamin D concentration was greater in epididymal fat than gastrocnemius muscle and liver, but 25(OH)D concentration was not significantly different between tissues. Soft tissues of rats fed crystalline vitamin D3 had higher concentrations of total vitamin D than those of rats fed yeast-derived vitamin D2, while total 25(OH)D concentrations were similar between vitamin D sources. This method is well suited to more complete studies of vitamin D bioavailability and metabolite tissue distribution.