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