Study On HPLC Analytical Method Of Axitinib And Its Residual In Cleaning Validation

Axitinib(AXI),a new type of biological targeted therapy for tumor drugs,is also a small molecule protein kinase inhibitor.There is little literature on its analytical method and no literature on the analysis method of its residue during the cleaning validation of production equipment,therefore,it is of great application value to study an analytical method of AXI and an analytical method of its residues during cleaning validation.Based on the review of civil and foreign literature,an analytical method of AXI and an analytical method of its residues during cleaning validation were studied.The details are as follows:1)The analytical methods of cleaning validation of pharmaceutical production equipment and AXI were introduced.2)The analytical method of AXI and chromatographic conditions were studied by high performance liquid chromatography(HPLC)external standard method.The results were as follows.Analytical instruments and equipment applied were as follows:the HPLC was Agilent 1260 HPLC,the UV detector was DAD,the HPLC column was Agilent Eclipse XDB C18(250 mm×4.6 mm×5?m).The preferred chromatographic conditions were as follows:the external standard was AXI reference substance,detection wavelength was 220 nm,sample size was 5?L,column temperature was45?,flow rate was 1.2 mL/min,mobile phases were ammonium dihydrogen phosphate buffer of pH 4.2(mobile phase A)and methanol(mobile phase B),gradient elution was applied(0 mins,A:B=50:50;20 mins,A:B=35:65;25 mins,A:B=10:90;25.1~30 mins,A:B=50:50).When the concentration of AXI was measured within the range of 198.5587~297.8381?g/mL,it had a good linearity that the correlation coefficient was R~2=0.9992.In addition,the stability accuracy,accuracy,precision(repeatability and reproducibility),specificity and robustness of the analytical method were also studied,and all could meet the requirements.3)An analytical method of residual AXI and its intermediates(AXI 30)during cleaning validation and chromatographic conditions were studied by HPLC external standard method.The results were as follows:Analytical instruments and equipment applied in the analytical method of residual AXI intermediate(AXI 30)were as follows:the HPLC UV detector were same as above,the HPLC column was Agilent Zorbax Eclipse Plus-C18(100 mm×4.6mm×3.5?m).The preferred chromatographic conditions were as follows:the external standard was AXI 30 reference substance,determine wavelength was 220 nm,sample size was 5?L,column temperature was 45?,flow rate was 1.2 mL/min,mobile phases were ammonium dihydrogen phosphate buffer of pH 4.2(mobile phase A)and methanol(mobile phase B),gradient elution was applied(0 mins,A:B=50:50;10 mins,A:B=43:57;15 mins,A:B=10:90;15.1~20 mins,A:B=50:50).The limit of quantitation of AXI 30 determined was 1.0068?g/mL,and when the concentration of AXI 30 was measured within the range of 1.0068~10.0675?g/mL,it had a good linearity that the correlation coefficient was R~2=0.9999.In addition,accuracy,precision and specificity of the analytical method were also studied,and all could meet the requirements.Analytical instruments and equipment applied in the analytical method of residual AXI were as follows:the HPLC UV detector were same as above,the HPLC column was Agilent Zorbax Eclipse Plus-C18(100 mm×4.6 mm×3.5?m).The preferred chromatographic conditions were as follows:the external standard was AXI reference substance,determine wavelength was 220 nm,sample size was 40?L,column temperature was 45?,flow rate was 1.2 mL/min,mobile phases were ammonium dihydrogen phosphate buffer of pH 4.2(mobile phase A)and methanol(mobile phase B),gradient elution was applied(0 mins,A:B=50:50;10 mins,A:B=43:57;15 mins,A:B=10:90;15.1~20 mins,A:B=50:50).The limit of quantitation of AXI determined was 0.0498?g/mL,and when the concentration of AXI was measured within the range of 0.0498~0.2491?g/mL?g/mL,it had a good linearity that the correlation coefficient was R~2=0.9998.In addition,accuracy,precision and specificity of the analytical method were also studied,and all could meet the requirements.The HPLC analytical methods of AXI and of its residue during cleaning validation in this study,which was not reviewed in literature,could accurately detect and quantify the residues of AXI and its intermediates.It is of great application value in pharmaceutical production and equipment cleaning validation.