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 mu L 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 x 2.1 mm, 1.7 mu m, 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 mu g/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.