Development and verification of a RP-HPLC method for determination of residual trifluoroacetic acid in human interferon alpha 2b stock solution
10.13200/j.cnki.cjb.004281
- VernacularTitle:人干扰素α2b原液中三氟乙酸残留量反相高效液相色谱检测方法的建立及验证
- Author:
SUN Ruixin
- Publication Type:Journal Article
- Keywords:
Reversed-phase high performance liquid chromatography(RP-HPLC);
Human interferon alpha 2b(hIFNα2b);
Residual trifluoroacetic acid
- From:
Chinese Journal of Biologicals
2024;37(8):996-1001
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveTo develop and verify a reversed-phase high performance liquid chromatography(RP-HPLC)method for the determination of residual amount of trifluoroacetic acid(TFA)in human interferon alpha 2b(hIFNα2b)stock solution,and preliminarily apply the method.MethodsThe conditions of liquid chromatography were as follows:ZORBAX300SB-C18 column(4. 6 mm × 250 mm,5 μm);mobile phase:0. 08% phosphoric acid(pH 3. 0)+ 5. 0% methanol solution;detection time:6 min;flow rate:0. 8 mL/min;column temperature:35 ℃；UV wavelength:210 nm;injection volume:20 μL;isocratic elution. The system adaptability,linearity,specificity,accuracy,precision,limit of quantitation(LOQ),limit of detection(LOD)and durability of the method were verified. The TFA residues in three batches of hIFNα2b stock solution samples were determined by the developed method and composite column Comixsil ACRP(100 mm × 4. 6 mm,5 μm).ResultsThe linearity of TFA was good in the range of 10-300 μg/mL(R2= 0. 998 4). The average spike recovery rate was 101. 8% and RSD was 2. 68%. The RSDs of repeatability and intermediate precision verification were 0. 46% and0. 45%,respectively. The LOQ was 10 μg/mL,and the LOD was 2 μg/mL. The slight fluctuation of pH of organic solvent,buffer salt and temperature of column under the specified conditions had no obvious influence on the detection results,and the peak area was affected when the detection wavelength changed. In addition,TFA residues were not detected in three batches of hIFNα2b stock solution by two methods.ConclusionThe developed RP-HPLC method has good system adaptability,specificity,accuracy,precision and durability,and can be used for the determination of TFA residues in hIFNα2b stock solution.
