Development and validation of a liquid chromatography-mass spectrometry method for the simultaneous quantification of artesunate and dihydroartemisinin in human plasma.

The present study describes the development and validation of a simple, sensitive, and specific liquid chromatography-mass spectrometry (LC-MS) analytical method used for the co-quantification of artesunate (ARS) and its active metabolite, dihydroartemisinin (DHA), in human plasma, using artemisinin (ARN) as an internal standard. The liquid-liquid extraction of samples was carried out using dichloromethane and tert.-methyl butyl ether (at a ratio of 8:2 v/v) and then evaporated to dryness by a stream of nitrogen gas at room temperature. Chromatographic separation and mass analysis were performed on the Agilent 1100 Series Liquid Chromatography/Mass Spectrometer Detector Trap system, using electrospray ionization as an interface. The stationary phase was an Elipse XDB-C18 column. The mobile phase contained acetonitrile and 0.003 M glacial acetic acid at a ratio of 62:38 (v/v) delivered at a flow rate of 0.5 ml per minute. Positive ion mode was selected to detect extracted ions at m/z 407 and 261 for ARS, at m/z 307 and 261 for DHA, and at m/z 305 for ARN. The retention times for alpha-DHA, ARS, beta-DHA, and ARN were 6.6, 8.0, 9.2, and 10.8 minutes, respectively, and the total chromatography run time was 12 minutes. The limit of detection (LOD) was 2 ng/ml while the limit of quantification (LOQ) was 10 ng/ml for both ARS and DHA. In order to address any complications caused by the spontaneous non-catalytic breakdown of ARS to DHA, two calibration curves were prepared separately for both analytes. These graphs were found to be linear over the range of 10 to 3,200 ng/ml (r2 > 0.99). The recoveries at concentrations of 100, 200, 400, and 800 ng/ml were 108, 106, 91, and 89%, respectively, for ARS and were 112, 95, 80, and 86%, respectively, for DHA. For ARN, the recoveries were 119, 119, and 90% for concentrations of 200, 400, and 800 ng/ml, respectively. ARS working solutions were not stable after two months of storage at 4 degrees C or after 21 days at room temperature. This newly developed LC-MS method was then applied for measuring of ARS and DHA concentrations in a healthy volunteer having received oral ARS at 200 mg once daily for 5 consecutive days. There was no decline in ARS concentration after repeated doses and the C(ss-max-average) for DHA was found to be 703 +/- 94 ng/ml at t(ss-max) of 2 h.