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

Abstract

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 °C, with or without preservative) up to 72 h before storage at −80 °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 °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 °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 °C if their collection is performed over 24 h.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-014-0764-5) contains supplementary material, which is available to authorized users.

Stability of targeted metabolite profiles of urine samples under different storage conditions

“…Other observed alterations in the metabolite profiles were derived under conditions that were not part of our study (e.g. 72 h or 12 weeks at 4 °C) (Lauridsen et al 2007; Roux et al 2015). Due to the comparably small number of measurements, our study is limited in statistical power.…”

“…Therefore, it is pivotal to determine the effects of pre-analytical sample handling, including storage conditions. Previous research on the pre-analytical effects on human urine samples was regarding non-targeted mass spectrometry (MS) or nuclear magnetic resonance (NMR) technology (Barton et al 2008; Bernini et al 2011; Budde et al 2016; Emwas et al 2015; Gika et al 2008; Lauridsen et al 2007; Roux et al 2015). …”

Preterm neonatal urinary renal developmental and acute kidney injury metabolomic profiling: an exploratory study

“…Samples were then stored at −80°C within 2–6 hours of collection until measurements by metabolomics analysis, similar to other metabolomics studies [10–13]. No preservatives were added to the urine samples as previous studies have shown urinary metabolites are stable for at least 4 weeks [14] and the use of preservatives when storing samples at −80°C have little to no impact on the resulting metabolomics profiles [15]. …”

Nutrimetabolomics: An Integrative Action for Metabolomic Analyses in Human Nutritional Studies

“…Saude and Sykes proposed that sodium azide should be added to all urine samples in order to stabilize metabolomic profiles acquired by NMR. However, other authors note that the use of this preservative is not mandatory if urine samples are correctly stored . In this line, Bernini et al .…”