Implementation of a generic liquid chromatographic method development workflow: Application to the analysis of phytocannabinoids and Cannabis sativa extracts

Abstract

A generic liquid chromatographic method development workflow was developed and successfully applied to the analysis of phytocannabinoids and Cannabis sativa extracts. Our method development procedure consists in four steps:i)The screening of primary parameters (i.e. stationary phase nature, organic modifier nature and approximate mobile phase pH) was carried out with a generic gradient on a short narrow bore column, using a system able to accommodate numerous solvents/buffers and columns. Instead of complete peak tracking, the number of peaks which can be separated was considered as a response at this level, to save time.ii)The optimization of secondary parameters (i.e. gradient conditions, mobile phase temperature and pH within a narrow range) requires only 12 initial experiments and the use of HPLC modeling software for data treatment. It allows to find out the best retention and selectivity for the selected compounds. Peak tracking was performed with a single quadruple mass detector in single ion recording mode, and UV detection (in a broad wavelength range).iii)The refinement step allows to further adjust column efficiency, by tuning column length and mobile phase flow rate. This can also be done virtually using HPLC modeling software.iv)The robustness testing step was also evaluated from a virtual experimental design. Success rate and regression coefficients were estimated in about 1 min, without the need to perform any real experiment.At the end, this method development workflow was performed in less than 4 days and minimizes the costs of the method development in liquid chromatography.