Optimization and Validation of a High-performance Liquid Chromatographic Method for Determination of Α- and Γ-tocopherol in Rat Plasma and Erythrocytes

An HPLC method for determination of αand γ-tocopherol in rat blood has been optimized and validated. The optimum means of separation and analysis of tocopherol homologs were selected on the basis of procedures found in the literature; documentary evidence is presented which shows precision and accuracy were satisfactory. Spectrofluorimetric detection was performed after separation on a Separon SGX NH2 analytical column with n-hexane–2-propanol, 97:3, as mobile phase; racemic tocol was used as internal standard. A suspension of erythrocytes was saponified and tocopherols were extracted in the presence of BHT, ascorbic acid, and pyrogallol. Plasma samples were extracted without saponification but with antioxidant protection. Validation revealed that intra-day and inter-day RSD in plasma were 0.78% and 4.15%, respectively, for α-tocopherol and 4.03% and 13.155%, respectively, for γ-tocopherol. Intra-day RSD for erythrocytes were 3.75% and 2. 89% for α-tocopherol and γ-tocopherol, respectively. Absolute percentage recovery from plasma and erythrocytes was 99.8 ± 7.5 and 94 ± 9.0, respectively, for α-tocopherol and 100.2 ± 7.5 and 96.9 ± 7.8, respectively, for γ-tocopherol. Limits of detection in plasma and erythrocytes were 0.005 and 0.002 μg mL, respectively, for α-tocopherol and 0.002 and 0.0015 μg mL, respectively, for γ-tocopherol. Limits of quantification in plasma and erythrocytes were 0.015 and 0.006 μg mL, respectively, for α-tocopherol and 0.006 and 0.0045 μg mL, respectively, for γ-tocopherol.