No 11. Reversed phase HPLC was used for the determination of paclitaxel and related compounds in both isocratic and gradient mode, using four different HPLC columns. With the selected acetonitrile - 0.05 M ammonium acetate gradient, 13 taxanes of a standard mixture were clearly separated on a phenyl column within 30 min. The system was tested with plant extracts and unknown taxanes were identified by LC-MS with an electrospray interface. A specialty ‘taxane’ column also achieved a baseline resolution of the 13 taxanes, but in 55 min. The developed methods were applied in the analysis of plant extracts.

No 12. Solid phase extraction (SPE) methods are studied for the sample pretreatment for HPLC analysis of Taxus cell suspension cultures. Various types of SPE materials were tested for the extraction of both the taxane standards and samples of various origin. Comparison between the different cartridges and the different elution solvents are made in terms of extraction recovery and sample clean-up. Selective elution of the taxanes is achieved by a gradient elution scheme.

No 13. Taxus baccata cell line has been characterized for growth, nutrient uptake, taxane production and for GGPP synthase activity. The enzyme is extracted by inclusion of non ionic detergent and its activity parallels taxane production for the first three days. The enzyme activity reaches its peak at day 3 of culture growth, while total taxane formation rises till day 9, when the maximum taxane accumulation in the cells is observed (~0.004% d.w.). After this day the taxane excretion in the medium increases, reaching its maximum at day 19.