An Ultraperformance LC-MS/MS Method for the Quantification of the Antimalarial Atovaquone in Plasma.

ABSTRACT

BACKGROUND: A primary modality in the treatment and prevention of malaria is the administration of antimalarial agents. Atovaquone (ATQ) has been used in single-drug and multidrug antimalarial applications; however, studies have demonstrated high interindividual drug variability. With the scarcity of analytical methodologies available in the literature, we have developed and optimized a rapid, ultraperformance (UP) LC-MS/MS method for the quantification of ATQ in human plasma. METHODS: ATQ was extracted from 25 µL K2-EDTA human plasma via protein precipitation with acetonitrile. Sample solutions were separated on a Synergi 2.5-µm Polar-RP 100A (100 × 2 mm) column. ATQ and its internal standard were detected over 1.3 min on an API 4000 mass analyzer using an electrospray ionization source operated in negative ionization and selected reaction monitoring modes. The method was validated in accordance with the Food and Drug Administration (FDA) Guidance for Industry Bioanalytical Method Validation recommendations. RESULTS: Owing to pharmacokinetic parameters associated with ATQ, 2 calibration curves were generated to quantify the drug across a dynamic concentration range. Two standard curves were established ranging from 250 to 5000 ng/mL and 5000 to 50000 ng/mL, respectively. QC levels for both lower and higher concentration ranges prepared at low (750 ng/mL, 12000 ng/mL), mid (2000 ng/mL, 22500 ng/mL), and high (4250 ng/mL, 42500 ng/mL) concentrations yielded interassay precision ≤9.1% and accuracy ≤±9.4%. Dilutional, stability, and matrix effects studies were also performed, and results were within acceptability limits. CONCLUSIONS: This work describes the development and analytical evaluation of a UPLC-MS/MS method for ATQ quantification in plasma. The described method is sufficiently sensitive for ATQ quantification in plasma to support preclinical and clinical trials.