UHPLC-MS/MS method optimisation and validation for Artesunate analysis in urine matrices

Background: Malaria is a significant health threat. Artesunate is an effective treatment for it, but because it binds strongly to haemoglobin, monitoring drug levels in urine is essential to determine its effective concentration. Objective: This study aims to optimise and validate the chromatographic conditions and sample preparation for quantifying artesunate in urine using Ultra-High-Performance Liquid Chromatography Tandem Mass Spectrometry (UHPLC-MS/MS). Method: Artesunate levels in urine were analysed via UHPLC-MS/MS, with artemisinin as the internal standard. Separation was achieved using a C18 column (Shim-pack XR-ODSIII, 50 mm × 2.0 mm, 1.6 μm) at a column temperature of 30°C. The optimal mobile phase consisted of methanol-0.3% formic acid in water (80:20, v/v), delivered at 0.3 mL/min under isocratic elution. Result: Detection utilised a triple quadrupole mass spectrometer in positive electrospray ionisation (ESI+) mode, monitoring transitions at m/z 407.15 → 261.10 for artesunate and m/z 305.00 → 151.10 for artemisinin. Sample preparation involved protein precipitation with 300 µL of methanol, followed by vertexing (one minute) and centrifugation (10,000 rpm, 15 minutes, 4°C). The method achieved a lower limit of quantification (LLOQ) of 2 ng/mL and demonstrated linearity across the range of 2–400 ng/mL. Conclusion: The method met 2022 EMA validation criteria, including selectivity, precision, linearity, recovery, accuracy, and stability.