Analysis of vitamin D₃-sulfate and 25-hydroxyvitamin D₃-sulfate in breastmilk by LC-MS/MS

Sulfated metabolites of vitamin D have been suggested to be in breastmilk, although current methods to measure sulfated vitamin D compounds in breastmilk by liquid chromatography-tandem mass spectrometry (LC-MS/MS) have not adequately accounted for increased aqueous solubility of these sulfated metabolites. The purpose of this study was to generate a method of LC-MS/MS for measuring vitamin D₃-3-sulfate (VitD₃-S) and 25-hydroxyvitamin D₃-3-sulfate (25OHD₃-S) specifically in human breastmilk. The resulting method uses methanol to precipitate protein and solid phase extraction to prepare the samples for LC-MS/MS. The limits of quantification for analytes in solvent were 0.23 ng/mL VitD₃-S and 0.2 ng/mL 25OHD₃-S. Various experiments observed concentrations ranging 0.53 to 1.7 ng/mL VitD₃-S and ≤ 0.29 ng/mL 25OHD₃-S. Both analytes were present in aqueous skim milk, demonstrating the enhanced aqueous solubility of these vitamin D sulfates. In conclusion, we describe an effective method for measuring VitD₃-S and 25OHD₃-S in breastmilk by LC-MS/MS.