Method development and erythrocytic uptake studies for artemisinin and select analogs using liquid chromatography - mass spectrometry

The goal of this project was to quantify the amount of artemisinin, a novel antimalarial endoperoxide that partitioned into both, uninfected as well as Plasmodium falciparum infected erythrocytes using a reliable, reproducible, and robust analytical technique. This assay would then be extended to the quantitation of select artemisinin analogs partitioned into the red blood cells during incubation. In order to optimize various parameters such as incubation time, concentration of artemisinin to be used, hematocrit selection etc., partition studies were first carried out using 14C-labeled artemisinin. At a hematocrit of 33%, the incubation time was optimized at 2 hours, and the extracellular concentrations of artemisinin used for uninfected and infected erythrocytes were 3.5 μM (1000 μg/ml) and 1.4 μM (400 μg/ml), respectively. However, this assay could not be used for other artemisinin analogs since it was not feasible to radiolabel all of them. Hence an alternative method had to be set up in order to carry out the uptake studies.; For this purpose, a high performance liquid chromatography method coupled with electrospray ionization mass spectrometry was developed and validated. Standard calibration curves constructed for these compounds were linear with a detection limit determined between 16 ng to 100 μg. The intra- and inter-day accuracy were found to be 104% and 100%, respectively with a 4% variation in precision. The validated assay was used to quantitate the uptake of artemisinin and its analogs into erythrocytes. The analyte was recovered from the red blood cells by solid-phase extraction and subsequently analyzed on a Waters Micromass ZQ Mass Spectrometer. The quantity of artemisinin taken up into the infected erythrocytes was almost twice as compared to the uninfected cells. These results correlated well with the data obtained using 14C-artemisinin. The drug partitioning results obtained were used to deduce a relationship, if any, between the biological activity of the analog and its uptake into the parasitized erythrocyte based on the physicochemical properties of the analog.