High-Throughput Analysis of Water-Soluble Choline and Related Metabolites in Human Milk

Abstract Objectives Choline and related metabolites play important roles in metabolic processes. Inadequate provision of these nutrients to the exclusively breast-fed infant can negatively impact its healthy growth and development. Methods We developed an UPLC-MS/MS method for analyzing choline (Cho), phospho-choline (PCho), glycerophospho-choline (GPCho), total choline (tCho = Cho + PCho + GPCho), betaine, carnitine, creatinine, dimethyl glycine (DMG), methionine, and trimethylamine N-oxide (TMAO) in human milk. Results Optimized results were obtained using a Phenomenex Luna Silica (2) column, 100 × 2 mm, 3 µm, and a gradient of 0.1% aqueous propionic acid (A) and acetonitrile (B) from 60% to 90% A over 2 min (Waters ACQUITY UPLC I-Class - SCIEX 4500TQ mass spectrometer). Sample preparation required only 5–10µL of milk, diluted 1:80 in methanol/water, 4:1, v/v, prior to analysis. Quantification was done using isotopically labeled internal standards and an external standard curve. Pooled human milk used for method validation showed recovery rates from 108–131% for all analytes, and an overall process efficiency from 54 to 114%. All standard curves revealed good linearity (r > 0.999). Milk from apparently healthy Brazilian mothers (1–120days pp) revealed large concentration ranges within and between analytes (IQR, mg/L): Cho 10.3, 20.3; GPcho 48.7, 101; PCho 134, 221; tCho 120, 166; betaine 0.25, 0.53; carnitine 3.35, 5.06; creatinine 2.92, 3.90; DMG 0.26, 0.54; methionine 0.47, 0.90. 63% of the milk samples reached the tCho value used for the Adequate Intake (1–6 mo). Conclusions Our newly implemented method enabled the simultaneous analysis of water-soluble forms of choline and related metabolites in human milk in minute amounts of sample, and requiring only minimalistic sample preparation. 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).