Project Summary Malaria remains one of the most challenging infectious diseases in the world causing roughly 200 million cases and half a million deaths annually. As the most vulnerable populations, pregnant women and children are recommended for preventive treatment. Our program has addressed fundamental questions as to how pregnancy and childhood development impact the pharmacokinetics (PK) and pharmacodynamics (PD) of dihydroartemisinin (DHA)-piperaquine (PQ), the preferred artemisinin-based combination therapy for chemoprevention. We demonstrated nearly a 40 percent reduction in PQ exposure in pregnant women and children compared to nonpregnant adults. However, considering PQ is highly protein bound, alteration of protein binding during pregnancy and childhood development may impact fraction of unbound PQ that is free to transverse biological membranes and exert pharmacological effect at target sites, therefore, dose adjustment should weigh in unbound PQ exposure. Although total PQ exposure has been well studied, unbound free PQ exposure remains to be unexplored. In this proposal, we will evaluate the unbound PQ pharmacokinetic exposure in pregnant women and children, leveraging resource from the existing clinical studies. We developed a sensitive method to measure free PQ at as low as 20 pg/mL and will explore two dimensional liquid chromatography (2D-LC) for unbound PQ separation and quantitation with a smaller plasma sample volume. Finally, we will also explore PK/PD modelling with unbound PQ and identify the unbound PQ concentrations associated with malaria protection. The knowledge gained on unbound PQ exposure is expected to optimize interpretation of total PQ exposure to inform treatment guidelines for pregnant women and children. The novel 2D-LC method, if succeeded, can be used as a general method for analysis of other unbound drugs.