Development and verification of a RP-HPLC method for determination of residual trifluoroacetic acid in human interferon alpha 2b stock solution
Objective To 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 solu-tion,and preliminarily apply the method.Methods The conditions of liquid chromatography were as follows:ZORBAX 300SB-C18 column(4.6 mm × 250 mm,5 μm);mobile phase:0.08％phosphoric acid(pH 3.0)+5.0％methanol solu-tion;detection time:6 min;flow rate:0.8 mL/min;column temperature:35 ℃;UV wavelength:210 nm;injection volume:20 pL;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).Results The 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％and 0.45％,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.Conclusion The 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.
Reversed-phase high performance liquid chromatography(RP-HPLC)Human interferon alpha 2b(hIFNα-2b)Residual trifluoroacetic acid
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