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

• Published in: Journal of AOAC International Vol. 102; no. 4; pp. 1059 - 1068
• Main Authors: Levêques, Antoine, Oberson, Jean-Marie, Tissot, Emeline Alexandra, Redeuil, Karine, Thakkar, Sagar K, Campos-Giménez, Esther
• Format: Journal Article
• Language: English
• Published: England AOAC International 01.07.2019
• Subjects: Analysis; Beta carotene; Breast feeding; Carotenoids; Carotenoids - analysis; Chromatography; Chromatography, Reverse-Phase - methods; Fluorescence; Humans; Liquid chromatography; Lycopene; Milk, Human - chemistry; Newborn infants; Nutrition; Nutritional requirements; Phytonadione; Reproducibility of Results; Retinoids; Tocopherols; Vitamin A; Vitamin A - analysis; Vitamin E; Vitamin E - analysis; Vitamin K - analysis; Vitamins
• ISSN: 1060-3271; 1944-7922
• DOI: 10.5740/jaoacint.19-0016

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. 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. 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. 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. 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.