Separation of tocopherols by nano-liquid chromatography

Nanoliquid chromatography (nano-LC) was used for the separation of tocopherols (δ-, γ-, α-TOH), α-tocopherol acetate (α-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 μm I.D. and 375 μm O.D. packed in our laboratory with RP 18 silica stationary phase of either 5- or 3-μm 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 R s and N/ m values were observed using 3-μm RP 18 particles. Experiments performed with capillaries packed with 3-μm RP 18 particles provided good limit of detection (LOD) and limit of quantification (LOQ) (for δ-, γ-TOH, α-TOH-Ac were 4 and 8 μg/ml, while for α-TOH were 6 and 10 μg/ml, respectively). The optimized method was applied to extracts of serum and pharmaceutical preparation containing α-TOH and α-TOH-Ac.