Optimization and Validation of the Reversed-Phase High-Performance Liquid Chromatography with Fluorescence Detection Method for the Separation of Tocopherol and Tocotrienol Isomers in Cereals, Employing a Novel Sorbent Material

The separation and determination of tocopherols (Ts) and tocotrienols (T3s) by reversed-phase high-performance liquid chromatography with fluorescence detection has been developed and validated after optimization of various chromatographic conditions and other experimental parameters. Analytes were separated on a PerfectSil Target ODS-3 (250 x 4.6 mm, 3 mu m) column filled with a novel sorbent material of ultrapure silica gel. The separation of Ts and T3s was optimized in terms of mobile-phase composition and column temperature on the basis of the best compromise among efficiency, resolution, and analysis time. Using a gradient elution of mobile phase composed of isopropanol/water and 7 degrees C column temperature, a satisfactory resolution was achieved within 62 min. For the quantitative determination, alpha-T acetate (50 mu g/mL) was used as the internal standard. Detection limits ranged from 0.27 mu g/mL (gamma-T) to 0.76 mu g/mL (gamma-T3). The validation of the method was examined performing intraday (n = 5) and interday (n = 3) assays and was found to be satisfactory, with high accuracy and precision results. Solid-phase extraction provided high relative extraction recoveries from cereal samples: 87.0% for gamma-T3 and 115.5% for delta-T. The method was successfully applied to cereals, such as durum wheat, bread wheat, rice, barley, oat, rye, and corn.