Quality by Design approach in the development of an UHPLC method for the analysis of selected polyphenols in Diospyros Kaki

The development of an UHPLC method for the analysis of selected polyphenols in Diospyros kaki, using a triple quadrupole mass spectrometer as detector, is presented. The optimization of the method was performed through the principles of Quality by Design, consisting in the implementation of tools like risk assessment and multivariate strategies. During the method scouting phase, different types of chromatographic columns were tested, and on the basis of the obtained selectivity an Acquity BEH UHPLC C18 was selected. The Critical Quality Attributes (CQA) were the critical resolutions of some isobaric compounds and analysis time. The Critical Process Parameters (CPP) were selected using the Ishikawa’s diagram and were related to the characteristics of both the mobile phase and the column: flow, temperature, initial mobile phase composition, formic acid percentage in eluents, type of organic eluent and gradient. The effects of the CPPs on the CQAs were evaluated by a multivariate strategy, at first using a screening phase and then Response Surface Methodology (RSM). In the screening phase the knowledge space was investigated by an asymmetric screening matrix. Graphic analysis of the effects made it possible to set the type of organic solvent (methanol) and the optimal value for the flow, and in addition to select new experimental domains for the other involved CPPs. In the RSM the postulated quadratic models were calculated by means of Doehlert Design and were graphically represented by contour plots. The search for the global optimum zone was performed by the sweet spot plots, which were analysed in order to identify the zones where the multidimensional combinations of the CPP values allowed the desired values for all the CQAs to be obtained (resolution values ≥1, analysis time ≤20 min). Hence, the Design Space was identified as the multidimensional combination of the CPPs that satisfied the requirements for the CQAs, with a probability π≥90%. The method was validated and applied to real samples obtained by the extraction of Diospyros kaki flesh from two different cultivars (Rojo Brillante and Kaki Tipo) and processed in different ways (fresh fruit without any treatment, treated with ethylene, treated with CO2). The results showed the remarkable concentration of some compounds (i.e. gallic acid, (+)-catechin, caffeic acid, p-coumaric acid, phloridzin), in agreement with literature data. Furthermore, compounds that have not been previously reported were found, including salycilic acid, luteolin, phloretin as well as ellagic acid, that was quantified in high concentration and the presence of which in persimmon was only reported occasionally in the peel of this fruit.