Metabolite identification in complex mixtures using nuclear magnetic resonance spectroscopy

Abstract

Metabolomics has become a major tool in the analysis of food samples and the investigation of the impact of food on human health. Food samples, and biofluids in which food-derived metabolites are found, are complex mixtures of metabolites. Metabolomics aims to capture the entire set of metabolites (small molecules) present in a sample, using methods such as mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. However, identification of those metabolites remains a challenging task, and this chapter describes these challenges in relation to analysis of complex mixtures using nuclear NMR spectroscopy. Major challenges include the large diversity in metabolites, the problem of spectral overlap, and the lack of available reference spectra. The identification of known metabolites (structural confirmation) using a combination of NMR spectroscopy methods and the importance of spectral databases and emerging software tools for structural confirmation are presented. The importance of NMR spectroscopy in structural elucidation of novel metabolites is also described, with the contribution of mass spectrometry and hyphenated systems highlighted. Finally, the debate on reporting standards for metabolite identifications and annotations to facilitate data sharing and the emerging scoring systems to communicate confidence in metabolite identifications are discussed. Without comprehensive metabolite identification, biological interpretation of metabolomics results may be misleading or incomplete and so understanding of how metabolites are identified and the confidence in those identifications is crucial.