Development and validation of UHPLC-MS/MS methods for determination of piperaquine in quantitative dried blood...

Piperaquine (PPQ) is an antimalarial drug used in combination with dihydroartemisinin. To support pharmacokinetic studies of PPQ in children and resource-limited settings, we developed assays to determine PPQ in dried blood samples (DBS) collected with Capitainer devices (10 μL blood) and in 10 μL plasma samples. Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was employed. DBS samples were soaked with 1% HCl to extract PPQ into solution followed by neutralization with NH₄OH and extraction with methyl acetate, while the plasma samples underwent liquid-liquid extraction. The organic layer was dried and reconstituted in acetonitrile-water-formic acid (50:50:0.5, v/v/v) before UHPLC-MS/MS analysis using a pentafluorophenyl reverse-phase column (2.1 × 30 mm, 1.8 μm). Atmospheric pressure chemical ionization in positive mode was used with multiple reaction monitoring of m/z 535 → 288 for PPQ and 541 → 294 for PPQ-d₆ as the internal standard. Both analytes eluted at 0.52 min, with a total run time of 2 min per sample. Calibration ranges were 1-500 ng/mL for DBS and 0.5-500 ng/mL for plasma. Recoveries were 54.4-59.0% for DBS and 69.3-82.9% for plasma, with negligible matrix effect and hematocrit effect. DBS samples were stable for at least 8 months at room temperature. Clinical validation with 46 pairs of simultaneously collected DBS and plasma samples demonstrated a strong linear relationship between PPQ concentrations in DBS and plasma (logC_DBS = 1.02 x logC_plasma + 1.01, R² = 0.919), with a median (IQR) DBS/plasma ratio of 2.84 (1.97-3.95). The assays will facilitate pharmacokinetic studies in children and resource-limited settings.