Development by a design of experiment approach of RP-HPLC methods for the analysis of plant secondary metabolites

High performance liquid chromatography in reversed phase separation mode, generally coupled to mass spectrometry (RP-HPLC-MS), is the technique of choice for the identification and quantification of bioactive plant secondary metabolites. The optimization of the HPLC methods is generally carried out by conventional trial-and-error approaches, requiring the screening of a variety of experimental conditions, which include column temperature, pH and composition of the mobile phases, in addition to shape and duration of the gradient elution program. This communication describes and discusses the application of computer-assisted development of RP-HPLC methods for the separation, identification and quantification of secondary metabolites extracted from plant tissues and plant-derived cosmetic and food product. The study has been conducted by a Design of Experiments (DoE) approach that allow the simultaneous optimization of gradient time (tG), column temperature (T) and binary eluent composition on the basis of retention times and peak areas of the analytes of interest, obtained in twelve different experiments. These experiments consist in the linear gradient separations of the investigated samples performed at two different gradient times and column temperatures, using the aqueous component of the mobile phase at three different pH values. The presentation describes the use of the above experimental data to construct 3-D resolution maps, which evaluate the influence of column temperature, mobile phase pH and composition, and gradient duration on the retention and resolution of a variety of analytes. In our study, 3-D resolution maps were constructed using either mixtures of standard phenolic compounds or samples of these compounds extracted from plants tissues or plant-derived products. The goal of our study was to model the variations that can help for the better selection of the mobile phase composition and gradient elution mode, in order to improve peak resolution and to reduce the analysis time, using a limited number of experiments and, consequently, reduced amounts of expensive and environmentally harmful chemicals. Excellent correlation between simulated and experimental separations of phenolic compounds are demonstrated. The application of the above DoE approach to the separation, identification and quantification of phenolic compounds occurring in a variety of plants and plant-derived cosmetic, food, and dietary supplement are illustrated and discussed.