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

## TL;DR

This study developed a method to measure 25-hydroxyvitamin D3 in rat brains and found that brain levels are much lower than serum levels and are affected by liver function.

## Contribution

A new LC/ESI-MS/MS method with derivatization was developed to quantify 25(OH)D3 in rat brain tissue.

## Key findings

- Brain 25(OH)D3 levels were about 1/100 of serum concentrations in normal rats.
- Bile duct ligation reduced both serum and brain 25(OH)D3 levels.
- The method showed good linearity and precision for measuring 25(OH)D3 in brain tissue.

## 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.