High-performance liquid chromatographic method development for determining plasma alfa-tocopherol in Thai smokers and non-smokers

Abstract

A simple and rapid reversed-phase high-performance liquid chromatographic method was developed to determine alpha-tocopherol in plasma. The mixture of acetonitrile and isopropanol was used to deproteinize plasma protein in only 100 micro litre sample prior to HPLC analysis having alpha-tocopheryl acetate as an internal standard (IS). The mobile phase composed of methanol and the effluent was quantitated at 292 nm. alpha-tocopherol and IS were eluted at 7.1 and 8.3 min, respectively. The concentrations of alpha-tocopherol were linear related to response in the range of 0-30 microgram/ml with the lowest limit of quantitation of 0.72 microgram/ml. The intra-day and inter-day accuracy and precision in term of %bias and %RSD were less than 15% and 10%, respectively. No endogenous interference was detected, indicating the specificity of the method. Plasma sample could be withstood at room temperature for 9 hours but at 47+-1 ํC for 15 days without any detectable deterioration. Plasma sample could be restored within three freeze-thaw cycles and the processed analyte was still stable within autosampler at 4 ํC till 6 hours. To analyze plasma samples from fifty-eight Thai males of both smoker (n=30) or non-smoker (n=28) volunteers using the developed method, the mean value of endogenous alpha-tocopherol in plasma was nonstatistically significant (p = 0.931) with the determined valued of 12.23 microgram/ml for smokers and 12.16 microgram/ml for non-smokers. In interpreting concentration ratio of alpha-tocopherol to triglyceride or to cholesterol plus triglyceride between smokers and non-smokers, the statistically significant difference was observed with the p-value of 0.010 and 0.034, respectively. Thus, the developed method was successfully proven to be utilized for endogenous alpha-tocopherol determination in human plasma. In considering endogenous alpha-tocopherol level as the indicator for smoking status, the cholesterol and triglyceride value would also play the role