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

Abstract A simple, sensitive and reproducible high-performance liquid chromatographic (HPLC) method with fluorescence detection was developed and validated for the simultaneous quantification of mixed tocotrienols (α-, γ- and δ-) in human plasma. The method involves a straightforward sample preparation step, where plasma samples are deproteinized using a mixture of acetonitrile and tetrahydrofuran (3:2, v/v), followed by direct injection into the HPLC system. Separation was achieved using a methanol mobile phase at a flow rate of 1.5 mL/min, with fluorescence detection at excitation and emission wavelengths of 296 nm and 330 nm, respectively. The method demonstrated excellent linearity over concentration ranges of 12.7 ng/mL to 2.54 μg/mL for α-tocotrienol, 19.2 ng/mL to 3.84 μg/mL for γ-tocotrienol and 4.6 ng/mL to 0.923 μg/mL for δ-tocotrienol, with quantification limits of 12.7 ng/mL, 19.2 ng/mL and 4.6 ng/mL, respectively. Recovery rates ranged from 85.0% to 111.2%, and both intra-day and inter-day precision showed relative standard deviations below 15%. The method was validated for accuracy, precision and stability under various conditions, including freeze–thaw cycles and long-term storage. This HPLC method offers a rapid, sensitive and reliable approach for quantifying tocotrienols in human plasma, making it suitable for preclinical and clinical studies, particularly in pharmacokinetic and bioavailability research.