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There are hundreds of nonprotein amino acids whose importance in food and biological matrices is still unknown. Many of these compounds can be found in food as products formed during the processing, as metabolic intermediates or because they are added to increase functional and nutritional properties of food. Moreover, this kind of amino acids have also demonstrated to play relevant roles in the pharmaceutical and clinical fields since they may be used therapeutically in the treatment of some pathologies and their levels may be related with some diseases. These facts imply that the analysis of nonprotein amino acids can be useful to obtain relevant information in the food and biological fields.