Computer-Assisted Approach for the Development of RP-HPLC Methods for the Separation and Quantification of Bioactive Plant Secondary Metabolites

Abstract

A variety of plant secondary metabolites have a remarkable position as bioactive components in medicinal plants and have evidenced to exhibit numerous biological activities and several health benefits against chronic and degenerative human diseases. Moreover, secondary metabolites occurring in edible plants form an integral part of human diet, contributing to the sensory properties of plant-based aliments and to their beneficial effects on human health. Numerous are the instrumental analytical separation techniques that are employed to identify and quantify the bioactive compounds occurring in medicinal plants and plant-derived food products. Among them, high performance liquid chromatography, mostly in reversed phase separation mode (RP-HPLC) and generally coupled to mass spectrometry (MS), is the technique of choice for the identification and quantification of plant secondary metabolites. The optimization of 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, composition of the mobile phases, as well as shape and duration of the gradient elution program (1). This communication describes the development of computer-assisted RP-HPLC methods for the separation, identification and quantification of phenolic compounds, which are a large class of plant secondary metabolites comprising a great number of heterogeneous structures that range from simple molecules to highly polymerized compounds.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 compounds performed at two different gradient times and column temperatures, using either the aqueous component of the mobile phase at three different pH values or a combination of two organic solvents at three different volume ratios as the gradient former.The RP-HPLC methods developed by the computer-assisted approach described in this paper were used for the separation and quantification of phenolic compounds occurring in fruits of Olea europaea, extra virgin olive oil (EVOO) and olive mill waste water (OMWW). .