Chemometrics-Assisted Optimization of Beta-/Gamma-Tocol Separation on a C₃₀ Stationary Phase in Reversed-Phase LC

This paper presents a chemometrics-assisted optimization study to improve the separation of tocopherol (-T) and tocotrienol (-TT) homologues on a C₃₀ stationary phase in reversed-phase HPLC. The HPLC settings were optimized using a central composite design and the response surface methodology. Flow rate, column temperature, and mobile phase composition were chosen as independent variables. Peak resolution (R_s), analysis time (t_R), and peak symmetries of the tocopherol isomers were chosen as response variables. Optimum performance in terms of R_s was obtained at a flow rate of 0.31 mL min⁻¹, a temperature of 8.70 °C, and % B content (methyl tert-butyl ether: methanol: water, 80:18:2, v/v/v) in the mobile phase of 38.12%. The analysis of variance and regression analysis gave adjusted R² values of 0.9841 for R_s, 0.9850 for t_R-(α-T), 0.9853 for t_R-(β-T), and 0.9204 for the peak symmetry of β-T. This confirms the good agreement of experimental data with predicted values. The close eluting peaks of β-/γ-tocol could be baseline separated at the optimized conditions at a minimized analysis time. Empirical second-order polynomial models were derived that gave statistically high significances (P < 0.0001). Hence, the models can be successfully employed to predict the optimum separation conditions of co-eluting peaks of β-/γ-tocols. The optimized method was successfully applied to determine the individual tocol homologues in various cold pressed edible oils. Total contents ranged from 15 to almost 2600 mg tocol kg⁻¹ oil.