LC-MS/MS method for the determination of two metabolites of tribendimidine, deacylated amidantel and its acetylated metabolite in plasma, blood and dried blood spots

Tribendimidine has emerged as potential alternative to praziquantel in the treatment of Opisthorchis viverrini infections. To support its clinical development program, a quantitative high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay was developed for tribendimidine's degradation product deacylated amidantel (dADT) and its acetylated metabolite adADT. Analytical sample preparation included protein precipitation for blood and plasma, and direct processing of dried blood spots (DBS). The analytes were detected by multiple reaction monitoring with electrospray ionization in the positive mode (dADT: 178.3→133.1m/z, adADT: 220.4→175.1m/z, tribendimidine 294.3→249.0m/z). A pentafluorophenyl (PFP) phase Kinetex analytical column (2.6μm, 100Å, 50mm×4.6mm) with a 6min lasting mobile phase gradient program of ammonium acetate and acetonitrile was applied. The method was validated with respect to precision, accuracy, linearity, sensitivity, and selectivity. The analytical range in plasma and blood was 1-1000ng/ml and in DBS 10-2000ng/ml (R(2)<0.99). Recoveries determined using four different human blood batches were in the range of 70-90%. Inter- and intra-assay accuracy and precision deviations were at least ≤12.2%. dADT and adADT were stable within the autosampler for 72h (10°C), for 4h at room temperature, for 3 month at -80°C, and after three freeze and thaw cycles. DBS samples should be stored at -20°C. The validation results demonstrated that the LC-MS/MS method is precise, accurate and selective and can be applied for pharmacokinetic studies with tribendimidine.