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
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
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.