AN INNOVATIVE METHOD FOR THE DETERMINATION OF VITAMIN D IN HUMAN MILK USING STABLE ISOTOPES

Background: Most extant methods for quantifying vitamin D (vit-D) metabolites in human milk (HM) are timeconsuming, cause losses during extraction and derivatization, and require large sample volume. Objective: To develop a simple, low-HM-volume, specific, and high-sensitive assay method for the quantification of vitD in HM using 6,9,9-deuterated-25-hydroxycholecalciferol (25OH-D3-6,19,19d3) as an internal standard (IS). Methods: Frozen HM samples were thawed and homogenized at 37ºC during 15min. Before extraction, 25OHD3-6,19,19d3 was added to 4mL of HM (10 ppb). Fat-soluble vitamins were extracted with 8mL of acetonitrile, and then reextracted twice with 12mL of hexane: dichloromethane (4:1). Speed Vac-dried vit D and 25-hydroxylated forms were derivatized by adding 600μL of acetonitrile containing 4phenyl-1,2,4-triazoline-3,5-dione (PTAD, 1mg/mL). The process was quenched with 400μL of water and filtered. 50μL of the sample were tested in liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) with a Kinetex (Phenomenex) C18-100X2, 1mm column. Results: The new method uses only 4mL of HM for vit-D extraction; drying of extracted lipids have been automatized, and derivatization carefully controlled to minimize vit-D losses, that otherwise are controlled by the addition of the IS. Separation and quantitation vit-D metabolites have been optimized using UHPLC followed by quadrupole MS-MS. This method has been validated using 40 Guatemalan HM samples; the median concentrations and (ranges) in IU/L for vit-D2, vitD3 and 25OH-D3 were, respectively: 12.8 (0-433.0); 15.5 (9.0221.0;), and 10.5 (0-44.0). Conclusion: This innovative method provides a simple, reliable, high-sensitive methodology for measurement of vitamin D and 25OH-D forms in HM.