Determination of 25‐Hydroxyvitamin D₃ in Rat Brain by Derivatization‐Assisted LC/ESI‐MS/MS

Recent studies have suggested that vitamin D deficiency may have relations with various neuropsychiatric diseases as well as bone diseases. However, the concentrations of vitamin D metabolites in the brain and the relationship between their brain and serum concentrations remain poorly understood. To answer these questions, we developed and validated an LC/ESI-MS/MS method for quantifying 25-hydroxyvitamin D₃ [25(OH)D₃], an established marker for assessing vitamin D sufficiency/deficiency, in the rat brain and compared the brain concentrations with the serum concentrations. To enhance the assay sensitivity and specificity, the 25(OH)D₃ was derivatized with 4-[4-(1-pipelidinyl)phenyl]-1,2,4-triazoline-3,5-dione (PIPTAD) after purification of the brain sample by a two-step solid-phase extraction. A good linearity was obtained within the range of 20-1000 pg/g tissue, and the intra-assay and interassay precision and accuracy were acceptable. In normal rats (n = 6), the brain 25(OH)D₃ concentrations ranged from 128 to 175 pg/g tissue, which were extremely low (approximately 1/100) compared to the serum concentrations. The bile duct ligation caused the decreased serum 25(OH)D₃ level, which produced the subsequent decreased brain 25(OH)D₃ level (44-79 pg/g, n = 6). These results strongly suggested that the serum 25(OH)D₃ concentration has a significant effect on its brain level.