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

Development and verification of a RP-HPLC method for determination of residual trifluoroacetic acid in human interferon alpha 2b stock solution

目的建立反相高效液相色谱(reversed-phase high performance liquid chromatography,RP-HPLC)法测定人干扰素α2b(human interferon alpha 2b,hIFNα2b)原液中三氟乙酸(trifluoroacetic acid,TFA)的残留量,并进行验证及初步应用。方法液相色谱条件为:ZORBAX 300SB-C18色谱柱(4.6 mm×250 mm,5μm);流动相0.08%磷酸(pH 3.0)+5.0%甲醇溶液;检测时间6 min;流速0.8 mL/min;柱温35℃;紫外检测器波长210 nm;上样量20μL;等度洗脱。对方法进行系统适应性、线性、专属性、准确性、精密性、定量限与检测限及耐用性验证。同时采用建立的方法和复合型色谱柱Comixsil ACRP(100 mm×4.6 mm,5μm)测定3批hIFNα2b原液样品的TFA残留量。结果TFA在10~300μg/mL范围内线性关系良好(R^(2)=0.9984);平均加标回收率为101.8%,RSD为2.68%;重复性和中间精密度验证的RSD分别为0.46%和0.45%;定量限为10μg/mL,检测限为2μg/mL;有机溶剂、缓冲盐pH与柱温箱温度在规定条件下轻微上下浮动对检测结果无明显影响,检测波长变化时峰面积会受到影响。采用2种方法检测3批hIFNα2b原液中TFA残留量,结果均未检出。结论建立的RP-HPLC法具有较好的系统适应性、专属性、准确性、精密性及耐用性,可用于hIFNα2b原液中TFA残留量的测定。

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 solution,and preliminarily apply the method.Methods The conditions of liquid chromatography were as follows:ZORBAX 300SB-C18 column(4.6 mm×250 mm,5μm);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:20μL;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,5μm).Results The linearity of TFA was good in the range of 10-300μg/mL(R^(2)=0.9984).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 O.45%,respectively.The LOQ was 10μg/mL,and the LOD was 2μg/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.