Human Milk Metabolic Profiling Using Biocrates MxP® Quant 500 Kit

Abstract Objectives Comprehensive metabolic profiling of human milk is a useful tool for examining its composition and relationship to maternal and infant metabolism and status. Methods A pooled milk sample was used to evaluate the Biocrates MxP® Quant 500 kit for human milk metabolomics (107 small molecules and 523 lipids) using an ABSciex 5500QTRAP mass-spectrometer in LC-MS/MS and flow injection analysis (FIA) mode. Additionally, milk from Brazilian mothers (A: 2–8, B: 28–50, C: 88–119d postpartum, ntotal = 25) was analyzed. Results 424 of the 630 assay metabolites were detected in the milkpool above the limit of quantitation (LOQ); 31 metabolites were below the lower limit of quantitation (LLOQ), while 7 were above the upper limit (ULOQ), mostly free fatty acids including arachidonic acid, docohexaenoic acid, and eicosapentaenoic acid. Bile acids were only detected below LLOQ. Concentrations measured in 5 different sample volumes (2–20μL, n = 10) showed satisfactory reproducibility (CV 3.7–20.0%) for 458 metabolites. Acceptable intraday variation (80–120%, 6 replicates) was achieved for 409 metabolites when spiking with 3 levels of QC-standards, but only for 127 metabolites after dilution (1:2 and 1:5). However, 396 metabolites revealed a good intraday variation when only considering the 1:2-dilution. Conclusions The MxP Quant® 500 kit was successfully employed for human milk providing data for over 400 metabolites in 10μL milk. and thus offers ability to use the same assay for both human plasma and milk, enhancing comparability of results by reducing analytical bias and increasing our ability to study milk as a biological system. Funding Sources Bill & Melinda Gates Foundation (OPP1148405), USDA/ARS Intramural Project (5306–51,530-019–00), and CNPq (Brazilian National Council for Science and Technology).