Objective To establish a high-performance liquid chromatography method for the content determination of related substances in Tenofovir Alafenamide Fumarate Tablets,and to establish a two-dimensional high-liquid chromatography-triple quadrupole mass spectrometry(2D-HPLC-MS/MS)method for the identification of the chemical structure of unknown hygrothermal degradation impurities.Methods For the content determination of related substances,the chromatography column was Waters XTerra MS C18 column(250 mm×4.6 mm,5 μm),the mobile phase was 0.02 mol/L phosphate(adjusted to pH 6.0 with 1 mol/L potassium hydroxide solution)-methanol(95∶5,V/V)-methanol(gradient elution),the flow rate was 1.0 mL/min,the detection wavelength was 260 nm,the column temperature was 35℃,and the injection volume was 20 μL.For the chemical structure identification,the chromatographic column was Aglient Zorbax Eclipse Plus C18 column(50 mm×3.0 mm,1.8 μm),the mobile phase was 0.1%formic acid aqueous solution-methanol(55∶45,V/V),the flow rate was 0.4 mL/min,the column temperature was 35℃,the positive ion mode was adopted with the electric spray ion source(ESI),the mass charge ratio(m/z)scanning range was 100-1 000,the pressure of atomized gas(nitrogen,N2)was 45 psi,the flow rate of dry gas(N2)was 6 L/min,the temperature was 350℃,the fragmentation voltage was 150 V,and the secondary mass spectrometry collision pyrolysis energy was 30 eV.The hygrothermal degradation impurities were identified based on retention time,molecular weight,and secondary fragments.Results The limits of quantification(LOQ)of tenofovir alafenamide and five degradation impurities A-E were 0.4,0.4,2.0,2.0,0.8,and 0.8 ng,respectively.The relative correction factors of impurities A-E were 0.61,0.70,3.16,0.75,and 0.85,respectively.The average recoveries of impurities A-E were 100.20%,95.23%,102.36%,94.48%,97.26%with RSDs of 0.50%,0.23%,0.89%,1.02%,and 1.67%(n = 9),respectively.The unknown hygrothermal degradation impurities were identified as impurity D lactose adducts and tenofovir alafenamide lactose adducts.Conclusion The method is specific,sensitive,accuracy,which is suitable for the content determination of related substances in Tenofovir Alafenamide Fumarate Tablets.Impurities such as lactose adducts in the tenofovir alafenamide can be quickly identified through characteristic fragment ions.
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