聚乙二醇化重组人干扰素α2b注射液中游离聚乙二醇含量的测定

Determination of free polyethylene glycol content in PEGylated recombinant human interferon α2b injection

目的建立测定聚乙二醇(polyethylene glycol,PEG)化重组人干扰素α2b(Peg-IFNα2b)注射液中游离PEG含量的高效液相色谱-蒸发光散射检测方法,并进行方法的验证。方法以Sepax Bio-C18为固定相,流动相A为0.1%三氟乙酸-水溶液,流动相B为0.1%三氟乙酸-90%乙腈-10%水溶液,梯度洗脱,蒸发光散射检测器检测不同浓度PEG对照品及Peg-IFNα2b注射液样品,建立高效液相色谱-蒸发光散射检测法,并对方法进行专属性、线性范围、检测限及加样回收率验证。结果高效液相色谱显示,PEG对照品与混有PEG对照品的Peg-IFNα2b注射液色谱峰分离度良好,且无其他色谱峰干扰;PEG对照品在6.25~100.00μg/mL范围内线性良好,R2=0.9903;最低检测限约为1.56μg/mL;低、中、高3个浓度的PEG对照品加样回收率分别为107.51%、103.52%和95.99%。结论建立的方法简单、准确、快速,可用于测定Peg-IFNα2b注射液中游离PEG含量。

Objective To develop and verify a method for determination of free polyethylene glycol content in PEGylated recombinant human interferonα2b(Peg-IFNα2b)injection by high performance liquid chromatography(HPLC)coupled with evaporative light scattering detection.Methods Sepax Bio-C18 with a column temperature of 25℃was adopted.The mobile phase A was 0.1%trifluoroacetic acid-water,while the mobile phase B was 0.1%trifluoroacetic acid-90%acetonitrile-10%water,with the gradient elution at a flow rate of 1.0 mL/min.The evaporative light scattering detector was used for determination of various concentrations of PEG control and Peg-IFNα2b injection.The developed HPLCevaporative light scattering detection was verified for specificity,linear range,detection limit and spike recovery.Results The free PEG showed a good separation with the Peg-IFNα2b injection mixed with PEG,without interference of other chromatographic peaks.The calibration curve of PEG control showed good linearity in the range of 6.25~100.00μg/mL,with a R2 value of 0.9903.The minimum detection limit was about 1.56μg/mL,while the spike recoveries of PEG at low,moderate and high concentrations were 107.51%,103.52%and 95.99%respectively.Conclusion The developed method was simple,accurate and rapid,which might be used for determination of free PEG content in Peg-IFNα2b injection.