Determination of γ- and α-tocopherols in human milk by a direct high-performance liquid chromatographic method with UV–vis detection and comparison with evaporative light scattering detection

A rapid direct method (Method I) for measuring γ- and α-tocopherols in human milk was developed and validated using reversed-phase high-performance liquid chromatography with ultraviolet/visible (UV–vis) detection. Human milk, with an internal standard (α-tocopherol acetate) added, was diluted in hexane. The chromatographic system consisted of a short column (50 mm × 2.1 mm I.D., 3 μm particle size) that allowed the separation of the γ- and α-tocopherols in less than 6 min. The new direct method (Method I) was compared with other methods. Method II (saponification with ultraviolet/visible detection) determined 24% and 22% less γ- and α-tocopherols, respectively. Method III (saponification with evaporative light scattering detection) gave the same values for α-tocopherol content as Method II. However, the amount of sample used in the application of Method III was higher than that used in Method II. Furthermore, Method I uses smaller amounts of solvents, and it is simpler and faster than Methods II or III. Only a small volume of sample is needed, which is an additional advantage for biological assays.