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

• Published in: Journal of agricultural and food chemistry 2012-03, Vol.60 (9), p.2076-2082
• Main Authors: Irakli, Maria N, Samanidou, Victoria F, Papadoyannis, Ioannis N
• Format: Article
• Language: English
• Subjects: acetates; barley; Biological and medical sciences; Cereal and baking product industries; Chromatography, High Pressure Liquid - methods; corn; detection limit; durum wheat; Edible Grain - chemistry; fluorescence; Food industries; Fundamental and applied biological sciences. Psychology; General aspects; Isomerism; isomers; isopropyl alcohol; Methods of analysis, processing and quality control, regulation, standards; oats; reversed-phase high performance liquid chromatography; rice; rye; Sensitivity and Specificity; silica gel; solid phase extraction; Spectrometry, Fluorescence; temperature; tocopherols; Tocopherols - isolation & purification; tocotrienols; Tocotrienols - isolation & purification
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/jf204470j

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 × 4.6 mm, 3 μm) 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 °C column temperature, a satisfactory resolution was achieved within 62 min. For the quantitative determination, α-T acetate (50 μg/mL) was used as the internal standard. Detection limits ranged from 0.27 μg/mL (γ-T) to 0.76 μg/mL (γ-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 γ-T3 and 115.5% for δ-T. The method was successfully applied to cereals, such as durum wheat, bread wheat, rice, barley, oat, rye, and corn.