Validated RP-HPLC Method for Simultaneous Determination of Tocopherols and Tocotrienols in Whole Grain Barley Using Matrix Solid-Phase Dispersion

Abstract

The vitamers of vitamin E such as α-, β-, γ-, and δ-tocotrienol and α-, β-, γ-, and δ- tocopherol are important phytochemical compounds with antioxidant activity and with potential benefits for human health. A high-performance liquid chromatography with fluorescence detection (HPLC-FLD) method was validated for their determination in whole grain barley samples. Tocol extraction was performed by an optimized matrix solid-phase dispersion (MSPD) protocol with neutral alumina (0.5 g) as the dispersion agent and methanol (5 mL) as the elution solvent. The analytical column was an Eclipse XDB C18 column (150 × 4.6 mm, 5 μm) and it was operated at room temperature. Mobile phase was consisted of methanol/acetonitrile/i-propanol (55:40:5 v/v %) and the elution was isocratic at a flow rate of 0.8 mL/min. Total analysis time was 12 min, and the detection of the tocols was performed with a fluorimetric detector where the excitation and emission wavelengths were set at 295 and 335 nm, respectively. Method validation was performed by means of intra-day (n = 5) and inter-day accuracy and precision (n = 8), sensitivity, and linearity. The linear regression coefficient (R ²) was higher than 0.99. The recoveries of the tocols from barley samples with the proposed extraction method were in an acceptable level (74–91 %) where the relative standard deviation ranged from 4.2 to 15.0 %. Limits of detection (LODs) and limits of quantification (LOQs) varied from 0.03 to 0.11 mg kg⁻¹ and 0.11 to 0.34 mg kg⁻¹, respectively.