High Performance Separation Techniques for Plant Secondary Metabolites: Fundamental and Practical Aspects

Plant secondary metabolites are organic compounds produced by organisms belonging to the vegetable kingdom besides the primary biosynthetic and metabolic routes. These phytochemicals, which are not directly related to the growth and differentiation of the various plant tissue and organ systems, play important ecological functions, such as defending the plant against pathogenic microorganisms and insect, resist to abiotic stresses or function as signalling molecules in inter-plant communication. Moreover, most secondary metabolites occurring as "non-nutritive" compounds in edible plants and in food of plant origin have found to play important roles in disease prevention and health-promoting effects, in addition to confer specific sensory characteristics. This communication discusses fundamental and practical aspects of reversed phase high performance liquid chromatography (RP-HPLC) employed for the separation, identification and quantification of plant secondary metabolites occurring in plants and food of vegetable origin. The presentation evaluates the influence of various operational parameters and experimental conditions affecting the separation performance of phenolic compounds, which are the most abundant class of plant secondary metabolites and, therefore, are widely distributed in the plant kingdom, form an integral part of human diet, and have a remarkable position as active components in functional foods and food supplements. Also discussed is the practical application of RP-HPLC to study the occurrence of secondary metabolites in selected edible plants as a function of their genetic differences and environmental grown conditions, as well as the determination of phenolic compounds in agro-food matrices during the transformation of raw ingredients into food and in the production of foods that have a potentially positive effect on health and disease prevention beyond basic nutrition. Most of the RP-HPLC methods developed for these studies used narrow-bore C-18 columns and a semimicro photodiode array detector (PDA) cell of 2.5 ?L, in conjunction with a mass spectrometer, equipped with an electrospray ionization source (ESI-MS). The C-18 narrow bore column was eluted by a multi-segment gradient of increasing concentration of acetonitrile in water-formic acid solution that has been optimized on the basis of the results of a study carried out to evaluate the influence of mobile phase composition and gradient shape on separation performance and detection sensitivity by ESI-MS. The identification of individual phenolic compounds was performed on the basis of their retention time and both UV-visible and mass spectra, acquired by PDA and ESI-MS, respectively.