Multiplex LC-MS/MS assay for simultaneous quantification of artesunate and its active metabolite dihydroartemisinin with pyronaridine, proguanil, cycloguanil, and clindamycin in pharmacokinetic studies.

backgroundMalaria, especially caused by Plasmodium falciparum, remains a major global health concern, particularly in sub-Saharan Africa. To combat rising drug resistance, innovative treatment approaches like triple artemisinin-based combination therapy (TACT) and multi-drug antimalarial combination therapies (MDACTs) are being explored.

methodsThis study introduces a robust and validated multiplex LC-MS/MS assay for the simultaneous quantification of key antimalarial drugs and their metabolites, including artesunate, dihydroartemisinin, pyronaridine, proguanil, cycloguanil, and clindamycin. Developed in accordance with EMA guidelines, the assay ensures high accuracy, sensitivity, and stability. Serum samples were prepared through a process of protein precipitation with acetonitrile, followed by the evaporation of the supernatant. The resulting residues were then reconstituted in a 50/50 mixture of aqueous 20 mM ammonium formate buffer and methanol for the analysis.

resultsThe assay achieves lower limits of quantifications of 1 ng/mL for proguanil, 0.2 ng/mL for cycloguanil, 1 ng/mL for artesunate, 4 ng/mL for dihydroartemisinin, 2 ng/mL for pyronaridine, and 5 ng/mL for clindamycin. The assay was successfully applied in a pharmacokinetic study conducted as part of a clinical trial in Gabon and Ghana, assessing novel drug combinations in both children and adults against a standard of care artemisinin-based combination therapy.

conclusionsThe developed assay can support the further clinical development of these TACTs and MDACTs, ultimately contributing to enhanced malaria treatment strategies.