Quantification of Vitamins A, E, and K and Carotenoids in Submilliliter Volumes of Human Milk

Background: Human milk is the optimal nutrition for all newborns in the first 6 months of life. In order to assess the nutritional needs of the breastfed infant, human milk is often characterized for multiple nutrients. Objective: To ensure that we minimize the volume of milk dedicated for research and optimize the number of nutrients characterized, we developed analytical methodologies for the determination of vitamins A (retinol), E (alpha and gamma tocopherol), K (phylloquinone and menaquinone-4), and five carotenoids (β-carotene, lycopene, β-cryptoxanthin, lutein, and zeaxanthin) using <1 mL human milk. Method: Vitamins E and K and carotenoids are simultaneously isolated from 750 μL milk by liquid–liquid extraction (LLE). Tocopherols and carotenoids are determined by normal-phase LC with fluorescence and ultraviolet detection respectively. Vitamin K is analyzed on the same extracts after resuspension and clean-up by reversed phase liquid chromatography coupled to tandem MS. The analysis of vitamin A involves saponification of 200 μL milk followed by LLE and determination by normal-phase LC with UV detection. Results: Full single-laboratory validation at four different concentration levels is presented. Recovery rates were within 90–105% in all except one case (retinol at 1.9 μg/mL, 88% recovery), with RSDs of repeatability and intermediate reproducibility below 10 and 15%, respectively for all the compounds. Conclusions and Highlights: To the best of best knowledge, this is the first report that allows for the characterization and quantification of vitamins A, E, and K and five carotenoids in <1 mL human milk.