Determination of vitamin E isomers in plasma using ultra performance liquid chromatography

A rapid method for quantification of vitamin E isomers (α-, β-, γ-, δ- tocopherol and tocotrienol) was developed by using ultra performance liquid chromatography (UPLC) from human plasma. This method involved liquid-liquid extraction of plasma sample to extract the lipophilic portion for injection into UPLC system. The UPLC system was equipped with fluorescent detector (296 nm excitation, 330 nm emissions) and C18 column with 1.7 μm particle size (2.1 x 110 mm column) for particle separation. The mobile phase comprises of 1.0 % of H2O and 99.0 % of methanol (HPLC grade) with flow rate adjusted at 0.4 ml min-1. Peak separations were accomplished after 3.50 minutes and 5 μl treated samples were required for injection. Results obtained showed that the UPLC system was able to separate, detect and quantitate six peaks, namely α-, γ-, δ- tocopherol and tocotrienol, respectively. However, it failed to separate and quantification both, β- tocopherol and tocotrienol due to low amount in nature. The calibration curve was linear for a concentration ranging from 1 μg ml-1 - 10 μg ml-1 of total vitamin E standard which r2 > 0.9996 for all isomers. The within day and between day coefficients of variation were less than 10.29 % (n=4) and 10.73 % (n=4) for all isomers. The accuracy of isomers quantification for 1 μg ml-1 (n=4) and 10 μg/ ml (n=4) vitamin E stocks were 93.52 % - 98.13 %. The within day and between day coefficients of variation for vitamin E standard were less than 7.59 % (n=4) and 9.74 % (n=4) for all isomers while for plasma sample were less than 10.29 % (n=4) and 10.73 % (n=4) for all isomers. The recovery rates for all isomer were ranging from 98.96 ± 7.22 and 107.50 ± 1.19 and the limit of detection was 2.53 ng. The present data suggested that this method required extremely short analysis time for example 2.50 min, required less samples for injection and excellent chromatographic reproducibility. (author)