The life sciences are currently being transformed by an unprecedented wave of developments in molecular analysis, which include important advances in instrumental analysis as well as biocomputing. In light of the central role played by metabolism in nutrition, metabolomics is rapidly being established as a key analytical tool in human nutritional studies. Consequently, an increasing number of nutritionists integrate metabolomics into their study designs. Within this dynamic landscape, the potential of nutritional metabolomics (nutrimetabolomics) to be translated into a science, which can impact on health policies, still needs to be realized. A key element to reach this goal is the ability of the research community to join, to collectively make the best use of the potential offered by nutritional metabolomics. This article, therefore, provides a methodological description of nutritional metabolomics that reflects on the state-of-the-art techniques used in the laboratories of the Food Biomarker Alliance (funded by the European Joint Programming Initiative "A Healthy Diet for a Healthy Life" (JPI HDHL)) as well as points of reflections to harmonize this field. It is not intended to be exhaustive but rather to present a pragmatic guidance on metabolomic methodologies, providing readers with useful "tips and tricks" along the analytical workflow.

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Food authenticity becomes a necessity for global food policies, since food placed in the market without fail has to be authentic. It has always been a challenge, since in the past minor components, called also markers, have been mainly monitored by chromatographic methods in order to authenticate the food. Nevertheless, nowadays, advanced analytical methods have allowed food fingerprints to be achieved. At the same time they have been also combined with chemometrics, which uses statistical methods in order to verify food and to provide maximum information by analysing chemical data. These sophisticated methods based on different separation techniques or stand alone have been recently coupled to high-resolution mass spectrometry (HRMS) in order to verify the authenticity of food. The new generation of HRMS detectors have experienced significant advances in resolving power, sensitivity, robustness, extended dynamic range, easier mass calibration and tandem mass capabilities, making HRMS more attractive and useful to the food metabolomics community, therefore becoming a reliable tool for food authenticity. The purpose of this review is to summarise and describe the most recent metabolomics approaches in the area of food metabolomics, and to discuss the strengths and drawbacks of the HRMS analytical platforms combined with chemometrics.

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The quantification of short-chain and medium-chain fatty acids is becoming more and more relevant in fecal and plasma samples due to their biological impact, which has been associated with colon rectal cancer and fiber consumption. For these reasons, a fast, cost-effective, and reproducible analytical method is highly required. In this research, a gas chromatography-mass spectrometry method based on full scan and multiple reaction monitoring (MRM) acquisition modes were optimized and validated for the analysis of short-chain and medium-chain fatty acids in three biological samples: human fecal water, fecal fermentation supernatants, and human plasma. Several extraction solvents (acidified water, diethyl ether, dichloromethane, ethyl acetate, and methyl tert-butyl ether (MTBE) were further evaluated, demonstrating that the latter was clearly the most suitable solvent with recoveries from 75.4 to 124.4% and coefficient of variations lower than 20%. The applicability of the GC-MS method was tested, for instance, acetic acid was quantified by using samples of plasma and feces from healthy donors at mean values of 66.9 μM and 24.5 mM, respectively. The optimized protocol could successfully find applications within multi-compartment human studies. In parallel, a second pilot experiment on fecal fermentation supernatants indicated that the proposed protocol is suitable to follow the formation of SCFAs during in vitro fermentation by the human gut microbiota. In summary, the present work provided an improved GC-MS method for precise and accurate quantification of SCFAs and MCFAs in human feces and plasma.

Tryptophan and tyrosine metabolism has a major effect on human health, and disorders have been associated with the development of several pathologies. Recently, gut microbial metabolism was found to be important for maintaining correct physiology. Here, we describe the development and validation of a UHPLC-ESI-MS/MS method for targeted quantification of 39 metabolites related to tryptophan and tyrosine metabolism, branched chain amino acids and gut-derived metabolites in human plasma and urine. Extraction from plasma was optimised using 96-well plates, shown to be effective in removing phospholipids. Urine was filtered and diluted ten-fold. Metabolites were separated with reverse phase chromatography and detected using triple quadrupole MS. Linear ranges (from ppb to ppm) and correlation coefficients (r2 > 0.990) were established for both matrices independently and the method was shown to be linear for all tested metabolites. At medium spiked concentration, recovery was over 80% in both matrices, while analytical precision was excellent (CV < 15%). Matrix effects were minimal and retention time stability was excellent. The applicability of the methods was tested on biological samples, and metabolite concentrations were found to be in agreement with available data. The method allows the analysis of up to 96 samples per day and was demonstrated to be stable for up to three weeks from acquisition.