Pharmacokinetic analysis
Self-reported adherence was assessed at each study visit, and empty pill strips were collected after the 2-week intervention phase. EDTA plasma samples were collected at the start of the intervention phase on day 0 (at 0 hours and at 6 hours after the first dose), day 7 (VPA only), day 14, and day 42. Plasma concentrations of VPA were analyzed by using a routinely implemented and validated assay (Multigent Valproic Acid Assays) at the Erasmus University Medical Center Pharmacy. For pyrimethamine measurements, we developed an assay (validated according to U.S. Food and Administration/European Medicines Agency guidelines) using the Waters Acquity UPLC-MS/MS. Samples (50 μl) were prepared and pumped into the column [Waters Acquity BEH C18 (1.7 μm, 2.1 × 100 mm), at 50°C]. A gradient (0.3 ml/min) with two eluents was used (A: 2 mM ammonium acetate + 0.1% formic acid in water; B: 2 mM ammonium acetate + 0.1% formic acid in methanol). The total run time was 4.2 min. Mass transitions used were mass/charge ratio (