Numerous metabolic profiling pipelines have been developed to characterize the composition of
human biofluids and tissues, the vast majority of these being for studies in adults. To accommodate
limited sample volume and to take into account the compositional differences between adult and
infant biofluids, we developed and optimized sample handling and analytical procedures for studying
urine from newborns. A robust pipeline for metabolic profiling using NMR spectroscopy was
established, encompassing sample collection, preparation, spectroscopic measurement and
computational analysis. Longitudinal samples were collected from five infants from birth until 14
months of age. Methods of extraction, effects of freezing and sample dilution were assessed and
urinary contaminants from breakdown of polymers in a range of diapers and cotton wool balls were
identified and compared, including propylene glycol, acrylic acid and tert-butanol. Finally,
assessment of urinary profiles obtained over the first few weeks of life revealed a dramatic change in composition, with concentrations of phenols, amino acids and betaine altering systematically over
the first few months of life. Therefore, neonatal samples require more stringent standardization of
experimental design, sample handling and analysis compared to adult samples in order to
accommodate the variability and limited sample volume.