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

Abstract

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.

The Effect of Pasteurization on the Antioxidant Properties of Human Milk: A Literature Review

“…Vitamin A: Vitamin A consists of provitamin A (carotenoids: α-carotene and β-carotene) and non-provitamin A (lutein, zeaxanthin, and lycopene). Provitamin A is the most important for vitamin A activity, however, non-provitamin A also has antioxidant properties [ 39 ]. Several studies investigated the effect of pasteurization on provitamin A.…”

Nitrate supplementation at two forage levels in dairy cows feeding: milk production and composition, fatty acid profiles, blood metabolites, ruminal fermentation, and hydrogen sink

“…The Lacti-Check ultrasound milk analyzer (Hopkinton, MA) was used to measure the fat and protein contents, while the SCC and MUN were determined using Fossomatic 90. The vitamins (A and E) were quantified using gas chromatography as described by Bae and Kratzsch (2018) and Levêques et al (2019). An additional milk sample (from each of the animals) was collected extracted, methylated and further analyzed for its FA content using gas chromatography as described by Klop et al (2016).…”

Quantitative analysis of vitamin D and its main metabolites in human milk by supercritical fluid chromatography coupled to tandem mass spectrometry

“…In parallel, 1 mL of the same non-spiked milk was extracted and the IS solution (25 μL) added just before PTAD derivatization. LLE and SN conditions were based on previously published methodologies [26,27] and adapted to the low volumes of sample available. Extraction solvent mixtures of slightly different polarities were also tested.…”