UPLC-MS/MS determination of acetylsalicylic acid,salicylic acid and isosorbide-5-mononitrate in dog plasma

Objective:To develop an UPLC-MS/MS method for determination of acetylsalicylic acid(ASA)and its metabolite(salicylic acid,SA),isosorbide-5-mononitrate(5-ISMN) in dog plasma.Methods:The dog plasma was extracted by ethyl acetate,and separated on a BEH C18(100 mm×2.1 mm,1.7 μm)column by gradient elution using acetonitrile-0.5% formic acid-5 mmol·L-1 ammonium acetate and acetonitrile-2 mmol·L-1 ammonium acetate as mobile phases respectively.A tandem mass spectrometer equipped with electrospray ionization source was adopted as the detector.Multiple reaction monitoring(MRM)transitions of m/z 178.9→136.9,m/z 136.9→64.9,m/z 249.6→58.9 and m/z 293.7→250.0 were measured in negative mode for ASA,SA,5-ISMN and internal standard,respectively.Results:The linear concentration ranges of the calibration curve for ASA,SA,5-ISMN were 2.0-2000.0 ng·mL-1,20.0-16000.0 ng·mL-1 and 9.6-9557.0 ng·mL-1,respectively;the lower limits of quantification was 2.0,20.0,9.6 ng·mL-1,respectively;the intra-day and inter-day relative standard deviations over the entire concentration range were less than 15%.Conclusion:The method is sensitive and suitable for pharmacokinetic study of ASA,SA and 5-ISMN in dog subjects.