Utilidad de un método que determina simultáneamente retinol y α-tocoferol en leche materna por cromatografía líquida de alta resolución

Abstract

Objective

To present the results obtained in the development of a simplified method, with a direct extraction of breast milk and detection by reverse phase high-performance liquid chromatography of retinol and α-tocopherol.

Materials and methods

An experimental 2x3 factorial design was used, with temperature variables, type of solvent and antioxidant effect, at 2 levels. Subsequent to the extraction, the samples were saponified with potassium hydroxide in methanol, then extracted with petroleum ether, and dried and reconstituted with ethanol. The chromatography conditions were established using microbondapak columns with UV/visible detector: methanol/water (96:4) mobile phase, wavelength for retinol 325, and 290 nm for α-tocopherol.

Results

The analytics parameters were: linearity; r² = 0.9955 (retinol), r² =0.9808 (α-tocopherol); detection and quantification limits: 1.1 μg/dL and 2.7 μg/dL (retinol), 0.9 μg/dL and 2.3 μg/dL (α-tocopherol). Method accuracy: the within-day relative standard deviation was 4.5%, and a between-day relative standard deviation of 4.8% for retinol, and 4.9% within-day and 4.1% between-day forα-tocopherol. For accuracy, recovery = 85.8 ± 7.8% for retinol and 98 ± 1.9% for α-tocopherol. Once validated, the method was applied to the quantification of retinol and α-tocopherol in 100 samples of breast milk. The values obtained were 63.9 ± 5.2 μg/dL for retinol and 359.0 ± 115.0 μg/dL for α-tocopherol.

Conclusion

The method is selective, linear, precise, sensitive and accurate. These characteristics together with their simplicity make the validated method convenient for the determination of retinol and α-tocopherol in breast milk.