Separation of tocopherols by nano-liquid chromatography

Nanoliquid chromatography (nano-LC) was used for the separation of tocopherols (delta-,gamma-, alfa-TOH), alfa-tocopherol acetate (alfa-TOH-Ac) and an antioxidant compound, namely butylated hydroxytoluene (BHT) used to prevent TOHs autoxidation. The separation was carried out in a fused silica capillary of 100 micron I.D. and 375 micron O.D. packed in our laboratory with RP18 silica stationary phase of either 5- or 3-micron diameter (23 cm long). The mobile phase was composed by mixtures of methanol (MeOH), acetonitrile (MeCN) and water. Typical analyses time for the separation of all the five components of the mixture were 6–9 min depending on the composition of the mobile phase. Efficiency and resolution were strongly influenced by the particle diameter and the highest Rs and N/m values were observed using 3 micron RP18 particles. Experiments performed with capillaries packed with 3 micron RP18 particles provided good limit of detection (LOD) and limit of quantification (LOQ) (for delta-, gamma-TOH, alfa-TOH-Ac were 4 and 8 microg/ml, while for alfa-TOH were 6 and 10 microg/ml, respectively). The optimized method was applied to extracts of serum and pharmaceutical preparation containing alfa-TOH and alfa-TOH-Ac.