Evaluating metabolic profiling of human milk using Biocrates MxP® QUANT 500 assay

Authors: HAMPEL, DANIEL - University Of California, Davis; Shahab-Ferdows, Setareh; KAC, GILBERTO - Universidade Federal Do Rio De Janeiro; Allen, Lindsay

Submitted to: Metabolites
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/24/2024
Publication Date: 1/3/2025
Citation: Hampel, D., Shahab-Ferdows, S., Kac, G., Allen, L.H. 2025. Evaluating metabolic profiling of human milk using Biocrates MxP® QUANT 500 assay. Metabolites. 15(1). Article 14. https://doi.org/10.3390/metabo15010014.
DOI: https://doi.org/10.3390/metabo15010014

Interpretive Summary: For gaining more insight into mother-milk-infant metabolic relationships examination of the human milk (HM) metabolomic profile could be very useful. Therefore, we tested the Biocrates MxP® Quant 500 assay for HM targeted metabolomics (106 small molecules, 524 lipids). We further used this assay to measure HM from apparently healthy Brazilian mothers (A: 2-8, B: 28-50, C: 88-119d postpartum, n(total)=25). Using a HM-pool we were able to observe 506 of the 630 detectable metabolites; 12 of them were found in concentrations above the upper limit of quantitation. Analyzing 5 different HM-pool volumes (2-20µL) showed that 458 metabolites were linear. Within-day accuracy of 80-120% was found for 469 metabolites after spiking with plasma controls, and for 342 after a 1:2-dilution. The results for HM from Brazilian mothers indicated concentration changes across the different lactation stages, e.g., significantly lower concentrations from colostrum to mature milk was observed for many flow-injection analytes and only few LC-MS metabolites, while increasing concentrations at the later lactation stages were mostly found for amino acids and related compounds. Thus, the MxP Quant® 500 assay is a useful application for HM metabolic profiling and allows the use of the same assay for HM and plasma, which enhances our ability to study milk as a biological system.

Technical Abstract: Metabolic profiling of human milk (HM) is indispensable for elucidating mother-milk-infant relationships. We evaluated the Biocrates MxP® Quant 500 assay for HM targeted metabolomics (106 small molecules, 524 lipids) and analyzed in a feasibility test HM from apparently healthy Brazilian mothers (A: 2-8, B: 28-50, C: 88-119d postpartum, n total=25). 506 of the 630 signatures were found above the limits of detection in a HM-pool (10µL) used for assay evaluation, 12 of them above the upper limit of quantitation. Analyzing 5 different HM-pool volumes (2-20µL) revealed acceptable linearity for 458 metabolites. Intraday accuracy of 80-120% was attained by 469 metabolites after spiking, and for 342 after a 1:2-dilution. Analyzing HM from Brazilian mothers revealed significantly lower concentrations from colostrum to mature milk for many FIA and only few LC-MS metabolites, including triglycerides, sphingomyelins, and phosphatidylcholines; higher concentrations at the later lactation stages were found predominantly for amino acid and related compounds. Thus, the MxP Quant® 500 assay is a useful tool for HM metabolic profiling, minimizing analytical bias between matrices, and enhancing our ability to study milk as a biological system.