Impact of collection conditions on the metabolite content of human urine samples as analyzed by liquid chromatography coupled to mass spectrometry and nuclear magnetic resonance spectroscopy

There is a lack of comprehensive studies documenting the impact of sample collection conditions on metabolic composition of human urine. To address this issue, two experiments were performed at a 3-month interval, in which midstream urine samples from healthy individuals were collected, pooled, divided into several aliquots and kept under specific conditions (room temperature, 4 A degrees C, with or without preservative) up to 72 h before storage at -80 A degrees C. Samples were analyzed by high-performance liquid chromatography coupled to high-resolution mass spectrometry and bacterial contamination was monitored by turbidimetry. Multivariate analyses showed that urinary metabolic fingerprints were affected by the presence of preservatives and also by storage at room temperature from 24 to 72 h, whereas no change was observed for urine samples stored at 4 A degrees C over a 72-h period. Investigations were then focused on 280 metabolites previously identified in urine: 19 of them were impacted by the kind of sample collection protocol in both experiments, including 12 metabolites affected by bacterial contamination and 7 exhibiting poor chemical stability. Finally, our results emphasize that the use of preservative prevents bacterial overgrowth, but does not avoid metabolite instability in solution, whereas storage at 4 A degrees C inhibits bacterial overgrowth at least over a 72-h period and slows the chemical degradation process. Consequently, and for further LC/MS analyses, human urine samples should be kept at 4 A degrees C if their collection is performed over 24 h.