Simple, Sensitive, and Reproducible High-Performance Liquid Chromatographic Method for Determination of Mixed Tocotrienol in Blood Plasma Using Fluorescent Detection

A simple, sensitive, and repeatable high-performance liquid chromatographic method using fluorescence detection was developed and validated for quantifying of mixed tocotrienols (α-, γ-, δ-) in human plasma. After deproteinization with a mixture of acetonitrile: tetrahydrofuran (3:2, v/v), the supernatant was directly injected into the HPLC system. The mobile phase was comprised of methanol. Analyses were conducted at a flow rate of 1.5ml/min with the detector operating at excitation wavelength of 296nm and emission wavelength of 330nm. The quantification limit is 4.6ng/ml for δ-T3, 19.2ng/ml for γ-T3, and 12.7ng/ml for α-T3, respectively. The mean absolute recovery values ranged from 85.0% to 111.2%, while the intra-day and inter-day relative standard deviations were all below 15%. The calibration curve was linear for α-, γ-, and δ-T3, at a concentration range of 12.7-2538.6ng/ml, 19.2-3839.3ng/ml, and 4.6-923.1ng/ml respectively. Moreover, only a small sample plasma volume (250µl) is required for the analysis, Overall, this method is validated with acceptable precision and accuracy, and can be used to analyse tocotrienol samples obtained from preclinical and clinical studies in a speedy and reproducible manner.