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Global metabolic profiling procedures for urine using UPLC–MS

Nature Protocols volume 5, pages 1005–1018 (2010)Cite this article

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Abstract

The production of 'global' metabolite profiles involves measuring low molecular-weight metabolites (<1 kDa) in complex biofluids/tissues to study perturbations in response to physiological challenges, toxic insults or disease processes. Information-rich analytical platforms, such as mass spectrometry (MS), are needed. Here we describe the application of ultra-performance liquid chromatography–MS (UPLC–MS) to urinary metabolite profiling, including sample preparation, stability/storage and the selection of chromatographic conditions that balance metabolome coverage, chromatographic resolution and throughput. We discuss quality control and metabolite identification, as well as provide details of multivariate data analysis approaches for analyzing such MS data. Using this protocol, the analysis of a sample set in 96-well plate format, would take ca. 30 h, including 1 h for system setup, 1–2 h for sample preparation, 24 h for UPLC–MS analysis and 1–2 h for initial data processing. The use of UPLC–MS for metabolic profiling in this way is not faster than the conventional HPLC-based methods but, because of improved chromatographic performance, provides superior metabolome coverage.

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Figure 1: Flow diagram of a typical MS-based metabolite profiling workflow.
Figure 2: A BPI UPLC–MS chromatogram of a urine sample run in positive ESI mode (top) and negative ESI mode (below).
Figure 3: MSE data from a urine sample.
Figure 4
Figure 5: A two-dimensional PCA scores plot (PC1 versus PC2) of human urine samples (blue) and QCs (red) obtained by UPLC–MS in positive ESI.
Figure 6: PCA time series plot showing the first PC component (t[1] versus samples in run order).

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Acknowledgements

The author E. Want acknowledges funding support from Waters Corporation. H. Gika acknowledges support from the European Committee through a European Reintegration Grant (ERG 202132).

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Authors and Affiliations

  1. Department of Surgery and Cancer, Biomolecular Medicine, Faculty of Medicine, Imperial College London, London, UK

    Elizabeth J Want, Elaine Holmes & Jeremy K Nicholson

  2. Department of Clinical Pharmacology, Drug Metabolism and Pharmacokinetics, AstraZeneca, Macclesfield, Cheshire, UK

    Ian D Wilson

  3. Department of Chemistry, Laboratory of Analytical Chemistry, Aristotle University of Thessaloniki, Thessaloniki, Greece

    Helen Gika & Georgios Theodoridis

  4. Pharmaceutical Business Operations, Waters Corporation, Milford, Massachusetts, USA

    Robert S Plumb & John Shockcor

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  1. Elizabeth J Want
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Want, E., Wilson, I., Gika, H. et al. Global metabolic profiling procedures for urine using UPLC–MS. Nat Protoc 5, 1005–1018 (2010). https://doi.org/10.1038/nprot.2010.50

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