Separation of tocopherols by nano-liquid chromatography

Fanali, Salvatore
Camera, Emanuela
Chankvetadze, Bezhan
D’Orazio, Giovanni
Quaglia, Maria Giovanna

^aIstituto di Metodologie Chimiche, Consiglio Nazionale delle Ricerche, P. O. Box 10, Area della Ricerca di Roma, Via Salaria Km 29,300, 00016 Monterotondo Scalo, Rome, Italy
^bLaboratorio di Fisiopatologia Cutanea, Istituto S. Gallicano (IRCCS), Via S. Gallicano 25/A, I-00153 Rome, Italy
^cMolecular Recognition and Separation Science Laboratory, School of Chemistry, Tbilisi State University, Chavchavadze Ave 1, 380028 Tbilisi, Georgia
^dDipartimento di Studi Farmaceutici, Università “La Sapienza”, P.le Aldo Moro, 5-00185 Rome, Italy

^*Corresponding author. Tel.: +39-06-90625328; fax: +39-06-90625849.

Received 29 June 2003; revised 10 October 2003; accepted 18 October 2003.

Journal of Pharmaceutical and Biomedical Analysis 35(2):p 331-337, April 2004. | DOI: 10.1016/S0731-7085(03)00646-0

Abstract

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 RP₁₈ 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 RP₁₈ particles. Experiments performed with capillaries packed with 3-μm RP₁₈ 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.

Abbreviations: TOHs: tocopherols; α-TOH-Ac: α-tocopherol acetate; Nano-LC: nano-liquid chromatography.