2012 Annual Report 1a.Objectives (from AD-416): Analyze multiple vitamins simultaneously in human milk. 1b.Approach (from AD-416): In Phase I of this project, we will develop GC-MS and other methods for measuring multiple micronutrients simultaneously in human milk, obtain normal values from United States and other samples and determine valid conditions for collecting milk from women taking micronutrient supplements. 3.Progress Report: This research contributes to objective 2A of the in-house parent project. Methods have been developed for simultaneous analysis of thiamin, riboflavin, flavin adenine dinucleotide, nicotinamide and pyridoxal in human milk. The protocol is very rapid and based on UPLC-MS/MS. A manuscript describing these methods has been published in J Chromatography B. The method has been applied to analysis of breast milk samples from a maternal micronutrient intervention study in Malawi (Log 39801) revealing that milk concentrations of thiamin, riboflavin and nicotinamide are only 5 to 35% of those reported in well-nourished populations. Maternal supplementation with the RDA for 6 months only increased these values to 20 to 40% of published values. A manuscript on these analyses is ready for submission. Work is ongoing to expand the number of micronutrients in breast milk that can be analyzed rapidly including phosphorylated forms of B vitamins, vitamin B12, retinol, and trace elements. This was performed in collaboration with a team in ICDDR, Bangladesh, to determine timing of breast milk collection to obtain an accurate measure of micronutrient secretion.