Rapid baseline-separation of all eight tocopherols and tocotrienols by reversed-phase liquid-chromatography with a solid-core pentafluorophenyl column and their sensitive quantification in plasma and liver

Of the eight natural vitamin E congeners (α-, β-, γ-, and δ-tocopherol and α-, β-, γ-, and δ-tocotrienol), the non-α-tocopherol congeners have unique biological properties that may contribute to human health. Their study in vivo has been complicated by the lack of a simple analytical method that completely resolves and sensitively detects all eight natural tocopherols and tocotrienols in biological matrices. We thus developed and validated (according to the FDA guidelines for bioanalytical method validation) the first reversed-phase liquid chromatographic method for the baseline-separation and quantification of all eight tocopherols and tocotrienols. Analytes were extracted from human plasma or mouse liver and separated on a Phenomenex Kinetex PFP column (2.6 μm, 150 × 4.6 mm) by elution with methanol:water (85:15, vol/vol) at a flow rate of 0.8 mL/min. The developed RP-LC method used a solid-core pentafluorophenyl stationary phase and achieved baseline separation of all eight vitamin E congeners within 15 min at a backpressure of 23 MPa, which is suitable for most conventional HPLC systems. The method was fast, linear, accurate, and precise with detection limits of 27–156 pg and good recoveries (82–122%) for all analytes. In conclusion, we developed and validated the first RP-LC method for baseline resolution of all eight tocopherols and tocotrienols extracted from plasma and liver, which should be useful for the quantification of individual vitamin E congeners in large epidemiological studies and randomized controlled trials.