Development and validation of a liquid chromatography and ion spray tandem mass spectrometry method for the quantification of artesunate, artemether, and their major metabolites dihydroartemisinin and dihydroartemisininglucuronide in sheep plasma

Recently, promising fasciocidal activities of artesunate and artemether were described in rats and sheep. Therefore, a liquid chromatography tandem mass spectrometry (LC25 MS/MS) method was developed to quantify artesunate, artemether, and their metabolites dihydroartemisinin and dihydroartemisinin-glucuronide in sheep plasma. Protein precipitation with methanol was used for sample workup. Reversed-phase high performance liquid chromatography (HPLC) was performed using an Atlantis C18 analytical column with a mobile phase gradient system of ammonium formate and acetonitrile. The analytes were detected by tandem mass spectrometry (MS/MS) using selected reaction monitoring (SRM) with electrospray ionization in the positive mode (transition m/z 267.4→163.0). The analytical range for dihydroartemisinin, dihydroartemisinin-glucuronide and artesunate was 10-1000 ng/ml and for artemether 90-3000 ng/ml with a lower limit of quantification of 10 and 90 ng/ml, respectively. Inter- and intra-day accuracy and precision deviations were less than 10%. Consistent relative recoveries (60-80%) were observed over the investigated calibration range for all analytes. All analytes were stable in the autosampler for at least 30 h (6°C) and after three freeze and thaw cycles. The validated method demonstrated that the LC-MS/MS method is precise, accurate, and selective and can be used for the determination of the artemisinins in sheep plasma. The method was applied successfully to determine the pharmacokinetic parameters of artesunate and its metabolites in plasma of intramuscularly treated sheep.