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 d