Development, validation and application of high performance liquid chromatography method for the determination of Artemisinin content in Artemisia annua l. extracts

Abstract

Malaria is a common and life-threatening parasitic disease in many tropical and subtropical areas especially the Africa region where about 90 % of the global malaria cases were recorded. In 2005, World Health Organization announced a switch in strategy to artemisinin combination therapy which is currently being used widely and is saving many lives. With the numerous potential uses of artemisinin and its derivatives, the future demand for the compound continues to rise. Chemical synthesis of artemisinin is an expensive and difficult multistep process that is accompanied by low yield thus Artemisia annua plant remains a major and economically viable commercial source of artemisinin. Objectives: The main objective of this study was to develop and validate a high performance liquid chromatography method that is suitable for analysis of artemisinin content in crude extracts of A. annua L. plant cultivated in different parts of Kenya. This is important since current methods used for analysis are only suitable for the analysis of artemisinin bulk material. Method: The content of artemisinin in the extracts was determined using high performance liquid chromatography method that was developed for the separation of the artemisinin from other components present in the crude extracts. The powdered leaves of the plant were subjected to various extraction conditions to determine the optimal extraction of artemisinin. The leaves of Artemisia annua L. were collected from various places in Kenya, namely; Malakisi, Emusaga, Kehancha, Kitengela and Kenyatta University. Results: The optimized chromatographic conditions for determining the content of artemisinin were a mobile phase consisting of acetonitrile-0.05 M monobasic potassium phosphate (40:60 % v/v), with 0.005 M hexanesulphonate, pH 6.0 delivered through the HPLC system at a flow rate of 1.0 mL/min. The stationary phase was a reverse phase octadecylsilane (C18) column maintained at a temperature of 40 °C. The diluent used to dissolve the extracts was acetonitrile. The method was validated for specificity, precision, and linearity according to the International Conference on Harmonization guidelines where the linear regression data analysis for the calibration plots showed good linear relationship in the concentration range of 25-120 % with respect to the peak area. Highest extraction content of artemisinin was found when the plant powder was subjected to cold maceration while stirring for 12 hours. The content of artemisinin was found to range from 0.68 % to 0.89 % with highest content found in the plant grown in Emusaga. Conclusion and recommendations: The method developed offers the advantage of being able to quantify artemisinin content in crude extracts of Artemisia annua L. plant using solvents and reagents that are easily available in the market. The HPLC method developed was precise and suitable for routine analysis of crude extracts of the plant. The study also showed that artemisinin content varies from region to region. This method was being reported for the first time and can be used for routine quality control analysis of the crude extracts of the plant.