Purification and characterization of Taxol and 10‐Deacetyl baccatin III from the bark, needles, and endophytes of <i>Taxus baccata</i> by preparative high‐performance liquid chromatography, ultra‐high‐performance liquid chromatography‐mass spectrometry, and nuclear magnetic resonance
Md. Nurullah, Zakiya Usmani, Sheeraz Ahmad, Bibhu Prasad Panda, Saima Amin, Showkat R. Mir
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.