Quantification of fat-soluble vitamins in human breast milk by liquid chromatography-tandem mass spectrometry

Sensitive quantification method for fat-soluble vitamins in human breast milk by liquid chromatography-tandem mass spectrometry was developed. Vitamins A, D and E were extracted from 10.0 mL of breast milk after saponifying by basic condition. Vitamin K derivatives were extracted from 3.0 mL of breast milk after lipase treatment. The corresponding stable isotope-labeled compounds were used as internal standards. For the determination of vitamin D compounds, derivatization with a Cookson-type reagent was performed. All fat-soluble vitamins were determined by liquid chromatography-tandem mass spectrometry in the positive ion mode. The detection limits of all analytes were 1-250 pg per 50 μL. The recoveries of fat-soluble vitamins were 91-105%. Inter-assay CV values of each vitamin were 1.9-11.9%. The mean concentrations of retinol, vitamin D₃, 25-hydroxyvitamin D₃, α-tocopherol, phylloquinone and menaquinone-4 were 0.455 μg/mL, 0.088 ng/mL, 0.081 ng/mL, 5.087 μg/mL, 3.771 ng/mL, and 1.795 ng/mL, respectively (n = 82). This method makes possible to determine fat-soluble vitamins with a wide range of polarities in human breast milk. The assay may be useful for large-scale studies.