Validation of a method for simultaneous determination of tocopherols and tocotrienols in cereals using Normal Phase HPLC

Abstract

A method for the simultaneous determination of tocols (α-, β-,γ-, δ-tocopherols and α-, β-,γ-, δ-tocotrienols) in cereals using Normal Phase-HPLC (Merck; column LiChroCART 250-4, Lichrospher Si 60, 5 μm) with fluorescent detection (Ex290 nm/Em350 nm) was validated. A mixture of hexane, ethyl acetate and acetic acid (97.3:1.8:0.9, v/v/v) was used as the eluent (1.6 ml/min). The analyses were performed after spectrophotometric standardization of standard ethanol solutions. The residual coefficients of variation for the regression equation y=ax2 + bx + c were 0.001-3.8%, with r2>0.999. The limit of quantitation was 0.05 mg/kg, the upper limit of determination range from 40 to 60 mg/kg, repeatability 6.2-11.8%, and reproducibility 8.7-18.4%. Uncertainty of the method (P≤0.05), estimated for the analyses performed in replicate, ranged from 28.9 to 85.9% for the tocol concentration of 0.05-0.80 μg/ml and from 14.7 to 27.3% for the concentrations above 0.80 μg/ml. Recovery was 93.3-103.1%. The analytical method described is precise, fast and inexpensive.