Development and Validation of a High-performance Liquid Chromatography-mass Spectroscopy Assay for Determination of Artesunate and Dihydroartemisinin in Human Plasma

A sensitive method has been developed and validated for the determination of artesunate and its active metabolite dihydroartemisinin (DHA) in human plasma using artemisinin as an internal standard. Solid phase extraction (SPE) using Oasis HLB extraction cartridges was used for sample preparation and analysis was performed on a Shimadzu LCMS-2010 in single ion monitoring positive mode using atmospheric pressure chemical ionization (APCI) as an interface. Positive ions were measured using extracted ion chromatogram mode. The extracted ion for artesunate, alpha- and beta-DHA was m/z 221 and for artemisinin was m/z 283. Chromatography was carried out using a Synergi Max-RP, 4 mu, 75 mm x 4.6 mm column using glacial acetic acid 0.1%, acetonitrile and methanol mixture (38:46.5:15.5) as a mobile phase delivered at a flow rate of 0.5 mL/min. The retention times of artesunate, alpha- and beta-DHA and artemisinin were 17.4, 11.8, 18.7 and 13.4 min, respectively, with a total run time of 21 min. The assay was linear over the range 1-3000 ng/mL for artesunate and DHA. The analysis of quality control samples for artesunate 50, 300, 1300 and 2600 ng/mL demonstrated excellent precision with relative standard deviation of 14.3, 11.3, 7.5 and 12.1%, respectively (n=5). Recoveries at concentration of 50, 300, 1300 and 2600 ng/mL were 75, 94.5, 74.3 and 75.5%, respectively; similar results were obtained for precision and recovery of DHA. This liquid chromatography-mass spectroscopy (LC-MS) method for the determination of artesunate and DHA in human plasma has superior specification for sensitivity, sample throughput and robustness than previous methods and can reliably quantitate concentrations of both (artesunate and DHA) compounds as low as 1 ng/mL.