人凝血因子Ⅷ制品中聚山梨酯80残留量高效液相色谱-电喷雾检测器检测方法的建立及验证  

Development and verification of high performance liquid chromatography coupled with charged aerosol detection for residual polysorbate 80 content in human coagulation factor Ⅷ products

作　　者：邵天舒[1] 丁锐[1] 周长明[1] 郭雷[1] SHAO Tian-shu;DING Rui;ZHOU Chang-ming;GUO Lei(Beijing Institute for Drug Controls NMPA Key Laboratory for Research and Evaluation of Generic Drugs,Beijing Key Laboratory of Analysis and Evaluation on Chinese Medicine,Beijing 102206,China)

机构地区：[1]北京市药品检验所国家药品监督管理局仿制药研究与评价重点实验室中药成分分析与生物评价北京市重点实验室,北京102206

出　　处：《中国生物制品学杂志》2021年第9期1105-1110,共6页Chinese Journal of Biologicals

基　　金：国家科技重大专项(2017zx09101001-007)。

摘　　要：目的建立人凝血因子Ⅷ制品中聚山梨酯80残留量的高效液相色谱(HPLC)-电喷雾检测器(charged aerosol detection,CAD)检测方法,并进行验证及初步应用。方法采用反相-离子交换混合色谱柱Oasis MAX(2.1 mm×20 mm,30μm),流动相A为含2%(V/V)甲酸的异丙醇∶水(20∶80)溶液,流动相B为含2%(V/V)甲酸的异丙醇溶液,流速为1.0 mL/min,柱温为30℃,进样量为30μL,梯度洗脱,检测人凝血因子Ⅷ中聚山梨酯80残留量。验证方法的系统适用性、线性范围、检测限、定量限、重复性、稳定性及准确性,并与HPLC-蒸发光散射检测器(evaporative light scattering detector,ELSD)检测法进行比较。应用建立的HPLC-CAD方法检测10批人凝血因子Ⅷ样品中聚山梨酯80残留量。结果聚山梨酯80与其他组分色谱峰分离度良好,无其他色谱峰干扰。经HPLC-CAD检测,聚山梨酯80标准品在12.8~256.3μg/mL浓度范围内线性关系良好,回归方程y=121.42 x+1.321 7,r=0.998 0;定量限约为3.20μg/mL,检测限约为1.07μg/mL;供试品溶液重复检测6次聚山梨酯80含量的RSD为0.86%;供试品溶液室温放置0、2、4、6、8及10 h聚山梨酯80含量的RSD为0.65%;浓度为32.8、41.0及49.2μg/mL的加标样品回收率为96.85%~101.49%;该方法的定量限、检测限、重复性、稳定性、准确性指标均优于HPLC-ELSD。10批样品中聚山梨酯80残留量在56.09~66.75μg/mL,均符合《中国药典》三部(2015版)要求(≤100μg/mL)。结论成功建立了检测人凝血因子Ⅷ制品中聚山梨酯80残留量的HPLC-CAD法,该方法重复性、稳定性及准确性良好,可用于检测人凝血因子Ⅷ制品中聚山梨酯80残留量。Objective To develop,verify and perliminarily apply a method for determination of residual polysorbate 80 content in human coagulation factor Ⅷ(FⅧ)products by high performance liquid chromatography(HPLC)coupled with charged aerosol detector(CAD).Methods Oasis MAX column(2.1 mm × 20 mm,30 μm)was adopted at a column temperature of 30 ℃.The mobile phase A was composed of isopropanol-water(20 ∶ 80) containing 2%(V/V) formic acid,while the mobile phase B was isopropanol containing 2%(V/V)formic acid,with gradient elution at a flow rate of1.0 mL/min.The sample load was 30 μL.The developed HPLC-CAD method was verified for systemic suitability,linear range,detection limit,quantitative limit,reproducibility,stability and accuracy,by which the determination result was compared with that by HPLC-ELSD(evaporative light scattering detector) method.Results The residual polysorbate 80 showed a good separation with other components of FⅧ products,without interference of any other peaks.The linear range of polysorbate 80 was 12.8~256.3 μg/mL,with a regression equation of y = 121.42 x + 1.321 7 and a correlation coefficients(r)of 0.998 0.The quantitative limit of the developed method was about 3.20 μg/mL,while the detection limit was about 1.07 μg/mL.The RSD of determination results of polysorbate 80 contents in six repeat test was 0.86%,while that the spike recoveries in samples after storage at room temperature for 0,2,4,6,8 and 10 h was 0.65%,of polysorbate 80 in samples at concentrations of 32.8,41.0 and 49.2 μg/mL were 96.85%~101.49%.The quantitative and detection limits of the developed method were lower that,while the reproducibility,stability and accuracy were superior to,those of HPLC-ELSD.The residual polysorbate 80 contents in ten batches of samples were 56.09~66.75 μg/mL,which met the requirements in Chinese Pharmacopeia(VolumeⅢ 2015 deition,not more than 100 μg/mL).Conclusion The HPLC-CAD method for residual polysorbate 80 content in F Ⅷ products was successfully developed,which was sensitive,re

关 键 词：人凝血因子Ⅷ 聚山梨酯80 高效液相色谱 电喷雾检测器 

分 类 号：R917[医药卫生—药物分析学]