Simultaneous Quantification of Artemether and its Active Metabolite Dihydroartemisinin in Human Plasma by Liquid Chromatography-Tandem Mass Spectrometry

A sensitive, selective and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of artemether (ART) and its active metabolite dihydroartemisinin (DHA) in human plasma using artemisinin (ARM) as the internal standard (IS). The analytes were extracted by liquid-liquid extraction and chromatographic separation was achieved on a reversed-phase Thermo HyPurity C18 (50 mm × 4.6 mm, 5 µm) column with a mobile phase consisting of 2 mM ammonium formate: methanol (15:85, v/v) in an isocratic mode at the flow rate of 0.600 mL/min. In order to quantify the analytes, triple quadrupole mass spectrometer equipped with electro spray ionization in the positive mode was used to obtain consistent precursor ammonium ion adducts [M + NH4]+ at m/z 316.5/163.5 for ART, 302.7/163.1 for DHA and 300.5/219.3 for ARM. The method was fully validated in the concentration range of 0.250 to 250 ng/mL using 50 µL human plasma. Intra- and inter-day accuracy and precision were within the acceptable limits of ±15 %. Stability experiments for ART and DHA were evaluated under different storage conditions. The assay was successfully applied to measure therapeutic plasma level of ART and DHA using COARTEM® (80 mg artemether and 480 mg lumefantrine) formulation in 12 healthy volunteers under fasting condition