高效液相色谱法检测聚乙二醇化重组人干扰素α2b注射液中N-羟基琥珀酰亚胺含量

Determination of N-hydroxy succinimide content in PEGylated recombinant human interferon α2b injection by high performance liquid chromatography

目的建立检测聚乙二醇化重组人干扰素α2b(PEGylated recombinant human interferonα2b,PEG-IFNα2b)注射液中N-羟基琥珀酰亚胺(N-hydroxy succinimide,NHS)含量的高效液相色谱(high performance liquid chromatography,HPLC)法。方法以TSK G3000SW(7.8 mm×300 mm,10μm,TOSOH)为色谱柱,用20 mmol/L PB(p H 7.0)-145 mmol/L Na Cl-5%异丙醇为流动相,流速为0.6 ml/min,检测波长为260 nm,建立检测PEG-IFNα2b注射液中NHS含量的HPLC方法,进行专属性、系统适应性、准确性验证,并确定该方法的线性范围、定量限和最低检测限。用建立的方法检测3批PEG-IFNα2b注射液中NHS的含量。结果该方法专属性、系统适应性及准确性良好;线性范围为0.00 977~2.50μg/ml(R2=0.999 985);最低定量限为0.002 4μg/ml(0.002%),最低检测限为0.001 2μg/ml(0.001%)。3批PEG-IFNα2b注射液样品中均未检出NHS,表明样品中NHS小于0.001%。结论建立的HPLC法准确、可靠,可用于PEG-IFNα2b注射液中NHS含量的测定。

Objective To develop a high performance liquid chromatography （HPLC） method for determination of Nhydroxy succinimide （NHS） content in PEGylated recombinant human interferon α2b （PEG-IFNα2b） injection.Methods TSK G3000SW （7.8 mm ＆#215; 300 mm,10 μm,TOSOH） column was adopted,using 20 mmol/L PB （pH 7.0）-145 mmol/L NaCl-5%（v/v） isopropanol as mobile phase at a flow rate of 0.6 ml/min.The detection wavelength was 260 nm.The developed HPLC method was verified for specificity,system suitability and accuracy,and determined for linear range,quantitative detection limit and minimum detection limit,and used for determination of NHS contents in three batches of PEG-IFNα2b injection.Results The developed HPLC method showed high specificity,system suitability and accuracy,of which the linear range was 0.009 77 ～ 2.50 μg/ml （R^2 =0.999 985）,and quantitative detection limit and minimum detection limit were 0.002 4 （0.002%） and 0.001 2 μg/ml （0.001%） respectively.No NHS was detected in three batches of PEG-IFNα2b injection,indicating that the NHS content was less than 0.001%.Conclusion The developed HPLC method was precise and reliable,which might be used for determination of NHS content in PEG-IFNα2b injection.