ORIGINAL PAPER
Validation of a method for simultaneous determination
of tocopherols and tocotrienols
in cereals using Normal Phase HPLC
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1
National Research Institute of Animal Production,
Central Laboratory,
32-084 Morawica, Poland
2
National Research Institute of Animal Production,
Department of Animal Nutrition and Feed Science,
32-083 Balice, Poland
Publication date: 2009-01-23
Corresponding author
R. Gąsior
National Research Institute of Animal Production,
Central Laboratory,
32-084 Morawica, Poland
J. Anim. Feed Sci. 2009;18(1):173-192
KEYWORDS
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.
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