Determination of sulfadoxine and pyrimethamine in microvolume human plasma using ultra high performance liquid chromatography-tandam mass spectrometry

To support the pharmacokinetic study of sulfadoxine (SD) and pyrimethamine (PM) in pregnant women and children, sensitive methods with small sample volume are desirable. Here we report a method to determine SD and PM with microvolume plasma samples: 5 µL plasma samples were cleaned up by protein precipitation with acetonitrile. The deuterated analytes were used as the internal standards. The samples after cleanup were injected onto an ACE Excel SuperC₁₈ column (50 × 2.1 mm, 1.7 μm, Hichrom Limited) connected to a Waters I class UPLC coupled with a Sciex Triple Quad 6500⁺ Mass Spectrometer and eluted with water and acetonitrile both containing 0.1% formic acid in a gradient mode at 0.8mL/min. Detection utilized ESI⁺ as the ion source and MRM as the quantification mode. The precursor-to-product ion transitions m/z 311→245 for SD and 249→233 for PM were selected for quantification. The ion transitions for the corresponding internal standards were 315→249 for SD-d₄ and 254→235 for PM-d₃. The simplest linear regression weighted by 1/x was used for the calibration curves. The calibration ranges were 1-200 µg/mL SD and 2 - 1000ng/mL PM. The mean (± standard deviation) recoveries were 94.3±3.2% (SD) and 97.0±1.5% (PM). The validated method was applied to analysis of 1719 clinical samples, demonstrating the method is suitable for the pharmacokinetic study with samples collected up to day 28 post-dose.