High throughput assay for the determination of piperaquine in plasma.

A high throughput assay for the determination of the antimalarial piperaquine in plasma has been developed and validated. The assay utilises 96-wellplate formats throughout the whole procedure, and easily enables a throughput of 192 samples a day using a single LC system. Buffer (pH 2.0; 0.05M) containing internal standard was added to 0.25mL plasma in a 96-wellplate (2mL wells). The samples were extracted on a MPC solid phase extraction deep well 96-wellplate (3M Empore). Piperaquine and internal standard were analysed by liquid chromatography with UV detection on a Chromolith Performance (100mm×4.6mm) column with a mobile phase containing acetonitrile–phosphate buffer (pH 2.5; 0.1M) (8:92, v/v) at a flow rate of 3.0mL/min. The within-day precisions for piperaquine were 3.3 and 2.3% at 40 and 1250ng/mL, respectively. The between-day precisions for piperaquine were 5.8 and 1.3% at 40 and 1250ng/mL, respectively. The total assay precisions using 29 replicates over 5 days were 6.7, 4.5 and 2.7% at 40, 200 and 1250ng/mL, respectively. The lower limit of quantification (LLOQ) and the limit of detection (LOD) were 10 and 5ng/mL, respectively using 0.25mL plasma. Using 1mL of plasma, it was possible to decrease LLOQ and LOD to 2.5 and 1.25ng/mL, respectively.