Journal of Pharmaceutical Research Vol. 8, No. 1, January 2009 : 16 ABSTRACT A high-performance liquid chromatographic (HPLC) method has been developed for the determination of Artemisinin in Artemisia annua Herb. The HPLC separation was carried out in isocratic mode using C18, sunfire column (4.6 x 250 mm, 5 m particle size) with a mobile phase composed of acetonitrile : water (85:15 v/v) at a flow rate of 1.0 ml/min. The detection was monitored at 216 nm. The method validation parameters showed good results for linearity, precision, accuracy, specificity, and in recovery studies. The calibration curve for Artemisinin was found linear from the range of 500 to 1200 g/ml. The interday and intraday studies (relative standard deviation) was obtained within the limits. The proposed HPLC method is precise, accurate and rapid for determination of Artemisinin in Artemisia annua Herb. Key words: Artemisinin; HPLC; Artemisia annua herb. Journal of Pharmaceutical Research Vol. 8, No. 1, January 2009 : 16-18. DEVELOPMENT OF A SIMPLE HPLC METHOD FOR THE QUANTITATION OF ARTEMISININ IN ARTEMISIA ANNUA HERB Saini PK 1 , Jain CL 2 , Singh RM 1* , Mathur SC 1 and Singh GN 1 1.Research and Development Division, Central Indian Pharmacopoeia Laboratory, Indian Pharmacopoeia Commission, Govt. of India, Ministry of Health & Family Welfare, Sector-23, Rajnagar, Ghaziabad-201002. 2. Department of Chemistry, M. M. H. College, Ghaziabad (U.P.) India. Received on : 13.08.2008 Revised : 05.01.09 Accepted : 09.01.09 *Correspondence : ipclab@vsnl.net, raman19662002@yahoo.co.in INTRODUCTION Artemisinin is chemically known as (3R,5aS,6 R,8aS,9R,12 S,12aR)-Octahydro-3,6,9- trimethyl-3,12-epoxy-12 H -pyrano[4.3- j ]-1,2- benzodioxepin-10(3H)-one. Artemisinin and Artemisia are official in Indian Pharmacopoeia 2007 1,2 and in International Pharmacopoeia 3 . It is used for the treatment of malarial infections. Artemisinin and its semisynthetic derivatives artemether and artesunate have been established as safe and effective antimalarials 4 . The toxicity of artemisinin drugs is much lower than that of quinine and its derivatives. Significant adverse effects or signs of toxicity have not been reported in human patients treated with therapeutic dosages. Several methods have been already reported by High Performance Liquid Chromatography using ELSD detector 5 , High Performance Thin Layer Chromatography 6 , Gas Chromatography - Mass Spectrometry 7 for the analysis of artemisinin and its analogues, but most of them are not widely adaptable to a large range of analogues. The objective of the present work was to develop a simple and reproducible HPLC method for the determination of Artemisinin in Artemisia annua herb. Review Article EXPERIMENTAL Materials and Methods All chemicals and reagents used were of HPLC grade. Acetonitrile was obtained from E. Merck, Mumbai and n-hexane was obtained from Qualigens Fine Chemicals, Mumbai. Standard Artemisinin and Artemisia annua herb were obtained from CIMAP, Lucknow, India. Instrument The instrument used for the study was a Waters High Performance Liquid Chromatography, Millennium 32 Software system equipped with pump 600, inline degasser, 717 plus Autosampler, 486 Tunable Absorbance Detector. A Waters Sunfire C18 column (250 4.6 mm), 5 m particle size was used as the stationary phase. Standard Preparation 10 mg of Artemisinin reference standard was dissolved in the mobile phase to produce 10 ml. Sample Preparation The collected Artemisia annua leaf were dried in shade, crushed into fine powder and the powder was passed through 80 mesh sieve, stored in airtight container at ambient condition. About 5 g of the powdered Artemisia annua was accurately weighed and refluxed with n-hexane for 6 hrs at 60–2 in an amber coloured round bottom flask. The contents obtained were cooled to room temperature and filtered through Whatman No. 41 and the filtrate was collected in 250-ml amber coloured volumetric flask. The marc was again refluxed with another 100 ml of n-hexane for 1 hour. The contents obtained were filtered and combined with the previous filtrate. The combined filtrate was evaporated to dryness on water-bath at 70C. The residue was dissolved and diluted with mobile phase to get final solutions of 1000 g/ml.