Optimization of high-efficient pre-column sample treatments and C18-UFLC method for selective quantification of selected chemical forms of tocopherol and tocotrienol in diverse foods

The improved pre-column methods followed by gradient elution C18-chromatography (C18-UFLC) with photodiode (DAD) and fluorescence (FLD) detection for analysis of tocotrienols (T3s), tocopherols (Ts), alpha-tocopheryl acetate and cholesterol in plant, algae and fish oils, milk and animal tissues have been described. C18-chromatography without saponification permitted quantification of T3s and Ts in oils. Quantification of tocols in milk involved saponification followed by C18-chromatography. beta-tocol and gamma-tocol were unseparated using C18-chromatography. Esterification of hydroxyl group of tocols with trifluoroacetic anhydride allows their satisfactory separation. The combination of esterification of tocols, C18-chromatography and DAD monitoring at 278 and 205 nm provide the suitable analytical tool for quantification of beta- and gamma-forms of tocols in biological samples. Our original C18-chromatographic methods are satisfactory precise, accurate, repeatable and offer low values of limit of detection (<10 ng/mL) and limit of quantification (<27 ng/mL) for assayed tocols and cholesterol.

Automated summary

This study describes the use of improved pre-column methods combined with gradient elution C18-chromatography and DAD and FLD detection for the analysis of tocotrienols, tocopherols, alpha-tocopheryl acetate, and cholesterol in various biological samples. The quantification of tocotrienols and tocopherols in oils can be performed using C18-chromatography without saponification. Saponification followed by C18-chromatography is required for the quantification of tocopherols in milk. Esterification of hydroxyl group of tocopherols allows their satisfactory separation. The combination of esterification, C18-chromatography, and DAD monitoring provides a suitable analytical tool for the quantification of beta- and gamma-forms of tocopherols in biological samples. The original C18-chromatographic methods used in this study are precise, accurate, repeatable, and have low limits of detection (<10 ng/mL) and quantification (<27 ng/mL) for the analyzed tocopherols and cholesterol.