Development and optimization of a method for the separation of platycosides in Platycodi Radix by comprehensive two-dimensional liquid chromatography with mass spectrometric detection

Comprehensive two-dimensional chromatography (LC × LC) using combinations of two columns (C18 × CN and C18 × NH2) was employed with electrospray (ESI) mass spectrometry to analyze platycosides from root extract. Based on the capability of the C18, CN and NH2 columns to separate the platycosides, the orthogonality in two-dimensional space according to each combination of columns was predicted from the correlation coefficients between the retention times of the 17 compounds separated by the independent CN and C18 columns, and NH2 and C18 columns. The expected distribution of the peaks was also compared with the two-dimensional plots obtained by practical separation in an LC × LC system. The increased peak capacities using C18 × NH2 allowed three minor components and five isomers of the platycosides to be newly separated, which were not identified with 1D-LC using the individual C18 column, whereas the combination of C18 × CN did not result in any improvement of the separation performance.