BACKGROUND: Different types of analytical methods, with different characteristics, are applied in metabolomics and lipidomics research and include untargeted, targeted and semi-targeted methods. Ultra High Performance Liquid Chromatography-Mass Spectrometry is one of the most frequently applied measurement instruments in metabolomics because of its ability to detect a large number of water-soluble and lipid metabolites over a wide range of concentrations in short analysis times. Methods applied for the detection and quantification of metabolites differ and can either report a (normalised) peak area or an absolute concentration. AIM OF REVIEW: In this tutorial we aim to (1) define similarities and differences between different analytical approaches applied in metabolomics and (2) define how amounts or absolute concentrations of endogenous metabolites can be determined together with the advantages and limitations of each approach in relation to the accuracy and precision when concentrations are reported. KEY SCIENTIFIC CONCEPTS OF REVIEW: The pre-analysis knowledge of metabolites to be targeted, the requirement for (normalised) peak responses or absolute concentrations to be reported and the number of metabolites to be reported define whether an untargeted, targeted or semi-targeted method is applied. Fully untargeted methods can only provide (normalised) peak responses and fold changes which can be reported even when the structural identity of the metabolite is not known. Targeted methods, where the analytes are known prior to the analysis, can also report fold changes. Semi-targeted methods apply a mix of characteristics of both untargeted and targeted assays. For the reporting of absolute concentrations of metabolites, the analytes are not only predefined but optimized analytical methods should be developed and validated for each analyte so that the accuracy and precision of concentration data collected for biological samples can be reported as fit for purpose and be reviewed by the scientific community.

BioMarin Pharmaceutical Inc San Rafael CA USA

Center for Proteomics and Metabolomics Leiden University Medical Centre Leiden The Netherlands

Computational and Systems Medicine Department of Metabolism Digestion and Reproduction Imperial College London London UK

Department of Biochemistry Cell and Systems Biology Centre for Metabolomics Research Institute of Systems Molecular and Integrative Biology University of Liverpool Liverpool L69 7ZB UK

Faculty of Medicine and Dentistry Institute of Molecular and Translational Medicine Palacký University Olomouc 779 00 Olomouc Czech Republic

National Center for Toxicological Research US Food and Drug Administration Jefferson AR 72079 USA

Office of Advanced Manufacturing National Institute of Standards and Technology Gaithersburg MD 20899 USA

Office of Weights and Measures National Institute of Standards and Technology Gaithersburg MD 20899 USA

School of Biosciences and Phenome Centre Birmingham University of Birmingham Birmingham UK

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