Abstract

Background: Most of the fat-soluble vitamins are not stable and decompose when exposed to light. Therefore, stability testing of the analytes in solutions and biological matrix is required during development and validation of analytical methods devoted to determination of these vitamins in biological fluids.

Objective: Determination of the stability of lipophilic vitamins, including retinol, α-tocopherol, β- carotene and 25-hydroxy-metabolites of vitamin D at all stages of the analysis.

Methods: Stability studies in methanol solutions and plasma samples were performed, including shortterm and long-term stability, freeze and thaw stability and autosampler test. For determination of vitamins in solutions, spectrophotometric method was applied. Analysis of vitamins in samples obtained following extraction of the analytes from plasma was performed using validated HPLC methods with UV detection.

Results: Short-term stability test showed that all studied vitamins were stable up to 4 hours when stored at +4°C and in ambient temperature without protection from light. During long-term stability test performed within 46 days at room temperature with no access to the light, retinol and β-carotene in solutions showed degradation while other analytes proved to be stable. No significant decomposition of vitamins in solutions stored at +4°C was observed during the above storage time. All the vitamins were stable in samples stored for 24 hours in autosampler and during three freeze-thaw cycles of plasma samples stored at -80°C.

Conclusion: In solutions, the most stable analyte was α-tocopherol, whereas the less stable was retinol. All vitamins were stable in plasma samples in the studied storage conditions. To avoid decomposition of these vitamins low temperature and protection from the light is recommended.

Keywords: Stability, HPLC-UV, fat-soluble vitamins, spectrophotometric method, methanol solutions, plasma samples.

Graphical Abstract