LC-MS/MS Methods For Determination Of A Novel Anti-HIV Tablet And Its Application To Pharmacokinetics Study In Beagle Dogs

Objective: A rapid,sensitive and convenient high-performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)method was developed for the determination of tenofovir,lamivudine and ACC007 in beagle dog plasma.The method was applied in the pharmacokinetics study of a novel anti-HIV tablet in healthy male beagle dogs after oral administration.Methods: API4000 triple-quadrupole mass spectrometer equipped with an electrospray ionization(ESI)source was used to develop a LC-MS/MS method for rapid determination of tenofovir,lamivudine and ACC007.Analytes were extracted from plasma using protein precipitation,tolbutamide was used as internal standard.Chromatographic separation was achieved on a Venusil MP C18 column(2.1 × 150 mm I.D.,5.0 μm)using A: 10 mmol·L-1 ammonium acetate containing 0.1 % FA,B: methanol as the mobile phase,which was delivered at a flow rate of 0.4 m L·min-1.The inject volume was 10.0 μL and a total chromatographic run time of 8 min was achieved.API4000 triple-quadrupole mass spectrometer equipped with an electrospray ionization(ESI)source was performed in the positive multiple reaction monitoring(MRM)mode using the transitions of m/z 288.1 → 176.2 for tenofovir,m/z 230.0 → 112.1 for lamivudine,m/z 326.1 → 298.3 for ACC007 and m/z 271.2 → 172.2 for tolbutamide(IS).Results: Calibration curve was linear over the concentration range of 20.00 ~ 2000 ng·m L-1 for tenofovir,lamivudine and ACC007.The lower limit of quantification was 20.00 ng·mL-1.The intra-and inter-day precision were less than 10.38 %,the accuracy was – 4.13 ~ 6.37 % for the three analytes,within the acceptable limit across the samples.The total chromatographic run time was 8 min.The method was successfully applied for pharmacokinetics study of the novel anti-HIV tablet ACC008 in male beagle dogs after oral administration.Conclusion: A successful LC-MS/MS method was established and completely validated for the determination of tenofovir,lamivudine and ACC007 in beagle dog plasma.The method was sensitive and selective and was successfully applied to the anti-HIV tablet ACC008 pharmacokinetics study in beagle dogs.