High-throughput analysis of water-soluble forms of choline and related metabolites in human milk by UPLC-MS/MS and its application

Authors: HAMPEL, DANIELA - University Of California, Davis; Shahab-Ferdows, Setti; NGUYEN, NGOC - University Of California, Davis; GILBERTO, KAC - Universidade Federal Do Rio De Janeiro; Allen, Lindsay - A

Submitted to: Frontiers in Nutrition
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/1/2020
Publication Date: 2/5/2021
Citation: Hampel, D., Shahab-Ferdows, S., Nguyen, N., Gilberto, K., Allen, L.H. 2021. High-throughput analysis of water-soluble forms of choline and related metabolites in human milk by UPLC-MS/MS and its application. Frontiers in Nutrition. 7. Article 604570. https://doi.org/10.3389/fnut.2020.604570.
DOI: https://doi.org/10.3389/fnut.2020.604570

Interpretive Summary: Choline and its related compounds are important for many processes in the human body and insufficient supply can negatively impact reproduction and fetal development. Choline status is mainly regulated by food consumption, and human milk is the only choline source for exclusively-breastfed infants. Additionally, mother and infant status and outcomes have been linked to milk choline concentrations. We have developed a method for the simultaneous analysis of choline, glycerophospho-choline, phospho-choline, betaine, carnitine, creatinine, dimethylglycine (DMG), methionine, and trimethylamine N-oxide (TMAO) in human milk using ultra-performance liquid-chromatography-tandem mass-spectrometry (UPLC-MS/MS). 10µL milk were mixed with water:methanol and filtered before analysis using an optimized gradient of 0.1% propionic acidaq and acetonitrile. Recovery of all compounds ranged from 108.0-130.9% (inter-day variation: 3.3-9.6%), and matrix effects (ME) from 54.1-114.3%. MEs were greater for carnitine, creatinine and TMAO at lower dilution (1:40, p<0.035 for all), indicating that the matrix is indeed affecting the recovery for some compounds. Milk from Brazilian women (2-8d, 28-50d, 88-119d postpartum) revealed increasing concentration during lactation for glycerophospho-choline, DMG, and methionine, and decreasing concentration for carnitine. Choline and phospho-choline revealed a negative relationship at all 3 collection time intervals. This method is suitable for rapid analysis of human milk water-soluble cholines and its related compounds, with minimal sample volumes and preparation.

Technical Abstract: Choline and related metabolites are key factors in many metabolic processes and insufficient supply can adversely affect reproduction and fetal development. Choline status is mainly regulated by intake, and human milk is the only choline source for exclusively-breastfed infants. Further, maternal status, geno- and phenotype, as well as infant outcomes have been related to milk choline concentrations. We have developed a method for the simultaneous analysis of choline, glycerophospho-choline, phospho-choline, betaine, carnitine, creatinine, dimethylglycine (DMG), methionine, and trimethylamine N-oxide (TMAO) in human milk using ultra-performance liquid-chromatography-tandem mass-spectrometry (UPLC-MS/MS). These analytes have milk concentrations ranging over 3 orders of magnitude. 10µL milk were diluted (1:80, v:v) in water:methanol and filtered prior to analysis with an optimized gradient of 0.1% propionic acidaq. and acetonitrile allowing efficient separation and removal of contaminants. Recovery rates ranged from 108.0-130.9% (inter-day variation: 3.3-9.6%), and matrix effects (ME) from 54.1-114.3%. MEs were greater for carnitine, creatinine and TMAO at lower dilution (1:40, p<0.035 for all), indicating concentration-dependent ion suppression. Milk from Brazilian women (2-8d, 28-50d, 88-119d postpartum) revealed increasing concentration throughout lactation for glycerophospho-choline, DMG, and methionine, while carnitine decreased. Choline and phospho-choline were negatively correlated consistently at all 3 collection time intervals. The method is suitable for rapid analysis of human milk water-soluble forms of choline and related metabolites, also in clinical settings, with minimal sample volumes and preparation.