Seasons Study of Four Important Taxanes and Purification of 10-Deacetylbaccatin III from the Needles of Taxus baccata L. by Two-Dimensional Liquid Chromatography

Simultaneous seasonal variation of four analogous taxanes, namely taxol (paclitaxel), cephalomannine, 10-deacetyl baccatin III (10-DAB), and baccatin III, from the needle of Iranian yew (Taxus baccata L.) has been monitored. Powdered samples of needles have been subjected to microwave-assisted extraction (MAE) and the extracted sample has been purified by liquid-liquid extraction (LLE) and analysis by reverse phase high performance liquid chromatography (RP-HPLC). These samples were identified by the LC-MS/MS system. The results indicated that taxane concentrations depend on seasonal and location. Analyses of these samples showed that 10-DAB has higher concentration than other taxanes and its highest concentration has been obtained in August. Thus, a heart cutting two-dimensional liquid chromatography was used for obtaining better resolution of taxanes. The first column in the analytical scale chromatography was a Eurospher-100 C8 and the second was Nucleosil-100 C18 which has been eluted at a flow rate of 1 mL min−1 with 70:30 v/v and 50:50 v/v water/acetonitrile, respectively. Optimized conditions in analytical chromatography have been extended to semipreparative scale in which the first column was a Eurospher-100 C8 and the second was a Eurospher-100 C18, both eluted with 70:30 v/v water/acetonitrile at a flow rate of 10 mL min−1. Detection has been carried out at a wavelength of 227 nm throughout the analysis in both scales. Final purity of the fraction containing 10-DAB, collected from the semipreparative column, is 82% after the first semipreparative run. Results have been confirmed by spiking of the standard 10-DAB sample and analysis by nuclear magnetic resonance (NMR).