高效液相色谱法测定重组人干扰素α1b滴眼液中苯扎氯铵的含量

Determination of benzalkonium chloride content in recombinant human interferon α1b eye drops by high performance liquid chromatography

目的采用高效液相色谱法(high performance liquid chromatography,HPLC)测定重组人干扰素α1b(recombi-nant human interferonα1b,rhIFNα1b)滴眼液中苯扎氯铵(benzalkonium chloride)的含量。方法采用HPLC法测定rhIFNα1b滴眼液中苯扎氯铵含量,色谱条件:流动相:乙腈:5 mmol/L醋酸铵[含1%三乙胺,用冰醋酸调节pH值至(5.0±0.2)]体积比为65∶35;流速:1.0 ml/min;检测波长:262 nm;进样量20μl;柱温:室温。对该方法进行验证,并应用该方法检测3批rhIFNα1b滴眼液中苯扎氯铵的含量。结果该方法检测苯扎氯铵专属性和系统适应性良好;最低检测限和定量限分别为0.53μg/ml和1.48μg/ml;苯扎氯铵在10～50μg/ml浓度范围内,标准曲线的线性关系良好,R2=0.999 1;连续6次进样,峰面积的RSD=1.78%;苯扎氯铵理论浓度为22、24、26μg/ml的加样回收率平均为97.90%,RSD=0.96%。3批rhIFNα1b滴眼液中苯扎氯铵的相对含量分别为19.27、19.89和19.76μg/ml,分别为标示量的96.34%、94.46%和98.80%。结论 HPLC法简便、灵敏、准确,可用于测定rhIFNα1b滴眼液中苯扎氯铵的含量。

Objective To determine the benzalkonium chloride content in recombinant human interferon α1b（rhIFNα1b）eye drops by high performance liquid chromatography（HPLC）.Methods The benzalkonium chloride content in rhIFNα1b eye drops was determined by HPLC.A Waters Symmetry C18 column was used with the mobile phase of V（A）∶ V（B）= 65 ∶ 35[A： acetonitrile;B： 5 mmol/L ammonium acetate containing 1% triethylamine,adjusted with acetic acid to pH（5.0 ± 0.2）]at a flow rate of 1.0 ml/min.The detection wavelength,sample load and column temperature were 262 nm,20 μl and room temperature,respectively.The method was verified,and used for determination of benzalkonium chloride contents in three batches of rhIFNα1b eye drops.Results The method showed good specificity and suitability in determination of benzalkonium chloride content,of which the minimum detection limit and minimum quantitation limit were 0.53 and 1.48 μg/ml respectively.The standard curve showed good linearity within a benzlkonium chloride concentration range of 10 - 50 μg/ml（R2 = 0.999 1）.The RSD of peak areas of six samples loaded were 1.78%.The mean recovery rate of samples at theoretical benzalkonium chloride concentrations of 22,24 and 26 μg/ml was 97.90%,with a RSD of 0.96%.The relative benzalkonium chloride contents in three batches of rIFNα1b eye drops were 19.27,19.89 and 19.76 μg/ml,which were 96.34%,94.46% and 98.80% of stated amounts,respectively.Conclusion The developed HPLC method was simple,sensitive and accurate,which might be used for determinatioin of benzalkonium chloride content in recombinant human interferon α1b eye drops.