Evaluation of Normal Phase High-Performance Liquid Chromatography for Analysis of Tocopherols

Song, Sean Mi; Jeong, Jin Yun; Lee, Kyoung Ryul

Abstract

BACKGROUND
Recently the tocopherol, which has been proven as an antioxidant, has also been found to possess non-antioxidant roles such as anti-inflammatory and chemopreventive effects, which has heightened the importance of rapid, accurate and reliable testing methods. We evaluated the performance of normal phase high-pressure liquid chromatography (NP-HPLC) for analysis of tocopherol in serum. METHODS: The chromatographic separation parameters, linearity, precision, accuracy and detection limit were evaluated. The analytical column was hypersil silica (4.6 mm*200 mm*5 micrometer, Thermo Electron Corporation, Keystone, PA, USA) and mobile phase comprised 2% (v/v) of isopropanol in n-hexane. Analyses were run at a flow rate of 0.7 mL/min at 295 nm. RESULTS: The run time of separation of tocopherol was completed in less than 15 minutes and its retention factor (0.61-2.13) and resolution including positional isomers ( beta- and gamma-tocopherol) were excellent. Their linearity was good (r>0.999). The coefficient of variation (CV) at medium and high concentrations did not exceed 15% and those at low concentrations did not exceed 20%. The range of its recovery was 91.4-107.8% and bias was within 15%. The detection limits of alpha-, beta-, gamma- and delta-tocopherol were 0.038, 0.064, 0.078 and 0.049, respectively. CONCLUSIONS: NP-HPLC provided a rapid, accurate and reliable performance for the separation of tocopherol and would be helpful as a routine method in the clinical laboratory.