Simple HPLC analysis of tocopherols and cholesterol from specimens of animal origin

Abstract

An improved saponification method followed by original isocratic high-performance liquid chromatography (HPLC) with photodiode detection (996 PAD, Waters) and/or fluorescence detection (474 Waters) for simultaneous analysis of cholesterol (CHOL) and δ-, α-, γ-tocopherols (δ-T, α-T and γ-T; forms of vitamin E) has been described. The method involved direct saponification of sample solutions flushed with a stream of argon, in the presence of vitamin C, followed by isocratic liquid chromatographic elution (Nova Pak C 18 column, 4 μm, 300 x 3.9 mm, I.D., Waters) and photodiode detection (UV) at 205 nm and/or fluorescence monitoring (λ ex /λ cm = 290/327 nm). Reversed-phase HPLC analyses have revealed that the optimum separation of CHOL and tocopherol from endogenous substances in biological samples can be obtained using the mobile phase containing 17% propan-2-ol and 83% of acetonitrile (v/v) at the flow rate of 1.5 mL min ―1 . Applying isocratic elution with UV monitoring at 205 nm and fluorescence detection, δ-T, α-T, γ-T and CHOL were eluted after 6.19 ± 0.09, 7.01 ± 0.08, 7.79 ± 0.08 and 14.7 ± 0.2 min, respectively. UV detection at 205 nm assured better detector responses for all tocopherols compared to other wavelengths. Detailed investigations have proven that alkaline saponification at 80°C for 15 min followed by isocratic chromatographic elution and UV and fluorescence detection enable simple and satisfactory simultaneous analysis of CHOL and δ-, α-, y-tocopherols in specimens of animal origin.