Purification and characterization of Taxol and 10‐Deacetyl baccatin III from the bark, needles, and endophytes of Taxus baccata by preparative high‐performance liquid chromatography, ultra‐high‐performance liquid chromatography‐mass spectrometry, and nuclear magnetic resonance

Abstract

Taxol and 10‐Deacetyl baccatin III are major taxanes in the bark, needles, and endophytes of Taxus baccata. The current study aimed to develop a process for their separation from different matrices. Crude taxoid was prepared by extraction of samples with methanol, followed by partitioning with dichloromethane and precipitation with hexane. Analytical high‐performance liquid chromatography involved isocratic elution on C18 column (4.6 × 250 mm, 5 μm) with methanol‐water (70:30 v/v) at a flow rate of 1 ml/min. Injection volume was 20 μl and detection was carried out at 227 nm. The content of Taxol and 10‐Deacetyl baccatin III in bark, needles and endophytic culture broth was 11.19 and 1.75 μg/mg; 11.19 and 1.75 μg/mg; and 2.80 and 0.22 μg/L, respectively. Preparative high‐performance liquid chromatography was done on C18 column (10 × 250 mm, 5 μm) at a flow rate of 10 ml/min. About 20 g crude taxoid was processed in < 3 h with a recovery of about 90% for both the analytes. The purity of recovered Taxol and 10‐Deacetyl baccatin III determined by ultra‐high‐performance liquid chromatography‐mass spectrometry was found to be 95.78 ± 3.63% and 99.72 ± 0.18%, respectively. The structure of recovered Taxol was confirmed by nuclear magnetic resonance. The method can find use in biotransformation studies.