Adsorption represents a critical challenge in the quantification of free 25-hydroxyvitamin D3.

Equilibrium dialysis followed by liquid chromatography-tandem mass spectrometry is considered the gold standard for the isolation and quantification of free hormones. However, this technique has not been described for the analysis of free 25-hydroxyvitamin D3. This study investigated analytical challenges, focusing on stability and adsorption of 25-hydroxyvitamin D3 in aqueous buffer environments, to be considered prior to method development of equilibrium dialysis for free vitamin D. Experiments revealed that 25-hydroxyvitamin D3 is highly prone to adsorption, and to a minor extent degradation, resulting in severe analyte loss already upon introduction of 25-hydroxyvitamin D3 into the aqueous environment, far exceeding observations in serum where protein binding provides significant protection. While chemical degradation only slightly contributes, strong surface adsorption accounts for the majority of 25-hydroxyvitamin D3 loss observed. In contrast, the addition of methanol to the buffer environment completely mitigated analyte loss, confirming the severe adsorption of 25-hydroxyvitamin D3 to recipient surfaces. However, incorporating methanol as a buffer component fundamentally altered experimental conditions, thereby potentially disrupting the physiological equilibrium between 25-hydroxyvitamin D3, vitamin D-binding protein, and albumin, and by extension the free fraction of vitamin D. Consequently, the large extent of, primarily, adsorption of 25-hydroxyvitamin D3 in aqueous environments represents an important consideration in setting up equilibrium dialysis experiments for free vitamin D. These findings additionally highlight a critical need for novel analytical strategies that prevent adsorption while maintaining physiological equilibrium in the assessment of free vitamin D.