Evaluation of RPLC stationary phases for tocopherol and tocotrienol positional isomer separation: method development and profiling

Reversed-phase separation of tocopherols (Ts) and tocotrienols (Ts) using C18 stationary phases results in the coelution of β and γ positional isomers, leading to identification errors. This study investigates the potential of alternative stationary phase chemistries to effectively resolve tocochromanols, specifically focusing on the critical pair of β and γ positional isomers. Initial screening of seven different stationary phases (C18, C18-PFP, C30, PFP, 5PYE, πNAP, and RP-Amide) was conducted. Linear solvent strength (LSS) studies were performed to assess the impact of the organic modifier (methanol) and temperature on the chromatographic performance parameters. Five columns were found to be suitable for the tocochromanol separation and two different chromatographical conditions per column were proposed. Elution order of tocochromanols was unique for 5PYE, πNAP and C30 columns in comparison to RP-Amide and PFP. Method development for the quantitative analysis of four tocopherol and four tocotrienol homologues was performed. The optimised method employed the RP-Amide (150 × 4.6 mm, 2.6 μm dp) superficially porous particle column, mobile phase of methanol:water of 92:8, v/v, with a flow rate of 1.0 mL/min, column oven temperature of 40 °C and fluorescence detection (λex 295 nm, λem 330 nm). The analysis run time was 10.5 min with 13.6 MPa back pressure. The method was validated and the obtained LOQs were found to be 1.30-3.13 μg/mL. The method developed was successfully applied for the determination of tocochromanols in twenty samples with unique tocochromanol profiles. Principal component analysis illustrated three distinct groups based on the tocochromanol profile.