Rapid and sensitive LC–MS/MS assay for sulfadoxine and pyrimethamine quantification in dried blood spots

Abstract Malaria is a mosquito-borne disease caused by parasites transmitted through the bites of infected female Anopheles mosquitoes. Malaria is primarily found in sub-Saharan Africa, where children under the age of 5 account for the majority of clinical cases and deaths. Sulfadoxine-Pyrimethamine is commonly used as Seasonal Malaria Chemoprevention during the high transmission season. In this study, we present a method for determining sulfadoxine and pyrimethamine in field-adapted Dried Blood Spots (DBS) samples on filter paper, using a simple and efficient extraction procedure. The method requires only 50 µl of capillary blood applied to filter paper, enabling a quantification range of 840 – 200,000 ng/ml for sulfadoxine and 4.4 – 1,000 ng/ml for pyrimethamine using LC–MS/MS. Analytes were separated using a Phenomenex Luna-CN 50×2 mm, 3 µm column, with a flow rate of 500 µl/min and a total analytical run time of 4.5 min per sample. The mobile phase consisted of 0.1% acetic acid in water and acetonitrile in a gradient flow. The method met all bioanalytical method validation requirements according to ICH M10 and FDA 2018 guidelines. It was successfully implemented for the analysis of 991 clinical DBS samples collected from children under the age of 5, on days 7 and day 28 after a single oral dose of sulfadoxine-pyrimethamine, as part of seasonal malaria chemoprevention.