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

Abstract

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).

Keywords:

• 10-Deacetyl baccatin III semipreparative
• Seasonal variation
• Semipreparative chromatography
• Taxol
• Taxus baccata L.
• Two-dimensional liquid chromatography

ACKNOWLEDGMENTS

This work has been supported by a grant from the Shahid Beheshti University Research Council and the authors gratefully acknowledge the support of this research by Medicinal Plants and Drugs Research Institute. We are also grateful to M. H. Mirjalili for gathering the plant materials.