Catharanthus Alkaloids in Cell Culture

The alkaloids of interest contained both an indole nucleus and a tertiary alicyclic amine. The buffer pH was adjusted to 6.5, since this is very near the pKa values of the amines 5.5 for vindoline and 6.8 for catharanthine. This pH also offered good separation between ajmalicine and catharanthine, the major alkaloids in our cell cultures, and ensured good column stability. 

Based on these electrochemical studies we developed a method for the quantitation of ajmalicine and catharanthine in cell cultures. These alkaloids were extracted from freeze-dried cells and purified by the solid-phase procedure described by Morris et al. (1985), except that ethanol was used as the extracting solvent instead of methanol. A dual-electrode coulometric cell was used in the screen mode. The potential of the first electode was set at +0.2 V (vs. Pd), which was at the base of catharanthine s voltammogram. The alkaloids were detected by the second electrode at +0.8 V, as this offered the best S/N ratio. Higher potentials led to lower S/N ratio, since the background current and noise started to increase exponentially above +0.85 V, due to the oxidation of water. The mobile phase was purified by a guard cell between the pump and injector. The guard cell operated at +0.8V. 

The detection limits of ajmalicine and catharanthine were 0.22 ng and 0.25 ng, respectively. This method was also selective, since usually no interfering peaks were observed in the chromatograms (Fig. 7). To insure reliability, a two-point calibration was performed daily, since the response varied, especially when starting after a weekend. Therefore it is recom- 

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Herein are described the principles, practice, and promise of LCEC in the determination of alkaloids in plant material. First the basic principles of LCEC relative to the determination of alkaloids are explained. Then the most recent methods applicable to LCEC detection of alkaloids in plant material are described, including our own work with Catharanthus alkaloids in cell cultures. Finally, we suggest applications for other alkaloids, which are based on published accounts of LCEC detection of alkaloids in body fluids and in other nonplant matrixes. 

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