Determination of gamma- and alpha-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 gamma- and alpha-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 (alpha-tocopherol acetate) added, was diluted in hexane. The chromatographic system consisted of a short column (50 mm x 2.1mm I.D., 3 microm particle size) that allowed the separation of the gamma- and alpha-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 gamma- and alpha-tocopherols, respectively. Method III (saponification with evaporative light scattering detection) gave the same values for alpha-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.
Department Nutrició i Bromatologia, Centre de Referència en Tecnologia dels Aliments (CeRTA), Facultat de Farmàcia, Universitat de Barcelona. Avda. Joan XXIII s/n, E-08028 Barcelona, Spain.
M
M
Romeu-Nadal
S
S
Morera-Pons
A I
AI
Castellote
M C
MC
López-Sabater
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D008027:Light; D008895:Milk, Human / Q000737:chemistry*; D012542:Scattering, Radiation; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D024502:alpha-Tocopherol / Q000032:analysis*; D024504:gamma-Tocopherol / Q000032:analysis*
D024504:gamma-Tocopherol; D024502:alpha-Tocopherol