Comprehensive Analysis of B-vitamins in Human Milk

Abstract Objectives B-vitamins are metabolic co-factors and an inadequate supply to the exclusively breast-fed infant can affect healthy growth and development. We developed a UPLC-MS/MS method for analyzing thiamin (T), thiamin-monophosphate, riboflavin, flavin adenine dinucleotide, flavin mononucleotide, nicotinamide, nicotinic acid, nicotinamide mononucleotide, nicotinamide dinucleotide, nicotinamide riboside, nudifloramide, pantothenic acid, pyridoxal, pyridoxal 5-phosphate, pyridoxamine, pyridoxine, 4-pyridoxic acid, biotin, 5-methyl-tetrahydrofuran (5MeTHF), and tryptophan in human milk. Methods Optimized results were obtained using a Phenomenex NX-C18 column, 250 × 3 mm, 3 µm, and a gradient of an aqueous solution of 10 mM ammonium formate + 0.05% ammonium hydroxide (A) and acetonitrile (B) from 99% to 5% A in a 12 min run (SCIEX ExionLC-AD -QTRAP6500+). Defatted and deproteinized milk (100 µL) was used and quantified by area ratio responses (external to internal standards). 50 milk samples from community-dwelling rural Bangladeshi mothers at 3.2 ± 0.2 months postpartum were analyzed and results were compared to the Adequate Intakes (AI) for infants 0–6 months. Results Analyte recoveries in pooled human milk ranged from 65–130% across vitamins with an overall process efficiency from 81–92% (T: 190%). Additional analytes were detectable but not yet quantifiable: nicotinamide dinucleotidephosphate, pyridoxamine 5-phosphate, 4-deoxypyridoxine, folic acid, and thiamin-diphosphate. 5MeTHF was detectable but given the sample preparation, only as a small concentration. Milk from Bangladeshi mothers had a large range of vitamin concentrations within and between analytes, including for samples meeting the AI. One sample (2%) reached the AI for vitamin B1, none for B2; 14% for niacin, 98% for pantothenic acid, 72% for B6, and 34% for biotin. Conclusions This method enables a comprehensive evaluation of milk B-vitamin concentrations to assess status and effects of maternal interventions on milk B-vitamins. Future application will be expanded to other matrices with further validation of the analytes currently only ascertainable qualitatively. Funding Sources Bill & Melinda Gates Foundation (OPP1148405), USDA/ARS Intramural Project (5306-51,530-019-00).