Multidimensional Liquid Chromatography for Separation of Cyclotides in Viola ignobilis

In this study, different columns, including C18, C8, and CN, were investigated for the analysis of cyclotides in Viola ignobilis. Because of the complexity of the cyclotide matrix in V. ignobilis and the presence of their different isomers, suitable separations could not be obtained by high-performance liquid chromatography (HPLC). Also, a miniaturized column with a powerful determination technique, such as nano-liquid chromatography-Fourier transform mass spectroscopy (nano-LC-FTMS), could not succeed in the identification of all the cyclotides. Thus, a multidimensional liquid chromatography (MDLC), in the heart-cutting and mixed-bed modes, has been developed to separate the cyclotides. In the heart-cutting MDLC, the C8-C18 and C8-CN conditions were investigated and a good resolution could be obtained for the isolation of a few specific peaks. In the mixed-bed mode, a C8-CN condition was investigated and good separation was obtained by a gradient condition. The results of these methods are compared.