Development, validation and preliminary application of highperformance liquid chromatography coupled with charged aerosol detector method for determination of polysorbate 80 in romiplostim
10.13200/j.cnki.cjb.004331
- VernacularTitle:罗米司亭中聚山梨酯80含量高效液相色谱-电喷雾检测器检测方法的建立、验证及初步应用
- Author:
GUO Ying
- Publication Type:Journal Article
- Keywords:
Romiplostim;
Recombinant protein drug;
High-performance liquid chromatography(HPLC);
Charged aerosol detector(CAD);
Polysorbate 80
- From:
Chinese Journal of Biologicals
2024;37(11):1361-1366
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop, validate and preliminarily apply a high-performance liquid chromatography(HPLC)-charged aerosol detector(CAD) method for the quantitative analysis of polysorbate 80 in recombinant protein drug romiplostim, in order to provide a reference for the quality control of recombinant protein drug preparations.Methods The HPLCCAD determination method for polysorbate 80 content was developed by screening the mobile phase and optimizing the elution gradient and CAD parameters. The specificity, repeatability and accuracy of the method were validated, and the limit of detection(LOD), limit of quantitation(LOQ) and linear range were determined. The content of polysorbate 80 in two batches of romiplostim samples was determined by using the developed method.Results HPLC-CAD conditions after method optimization were as follows: Acclaim Surfactant Plus column(150 mm × 4. 6 mm, 3 μm) was adopted, and the column temperature was 25 ℃. The 0. 1% formic acid in water and 0. 1% formic acid in isopropanol were used as mobile phase A and B respectively, with a flow rate of 0. 6 mL/min and a gradient elution(0 min 80% A, 1. 8 min 80% A, 2. 0 min66. 5% A, 3. 0 min 66. 5% A, 3. 5 min 0% A, 8. 5 min 0% A, 9. 0 min 80% A, 15. 0 min 80% A). The injection volume was10 μL. The evaporative temperature for CAD was 50 ℃ and the power function value(PFV) was 1. 25. There was no interference of other chromatographic peaks in the sample matrix and blank solution in this method. The RSDs of peak area for repeatability at low(10 mg/L), medium(50 mg/L) and high(80 mg/L) concentrations were 4. 32%, 1. 97% and 3. 46%, and the mean spike recoveries were 95. 7%, 99. 0% and 98. 9%, respectively. Polysorbate 80 had a good linear relationship in the concentration range of 5. 0-100. 0 mg/L(R2= 1. 000 0), and the LOD and LOQ in the method were 1. 0 and 2. 0 mg/L respectively. The content of polysorbate 80 detected by the developed method was 33. 20 and 32. 28 mg/L in two batches of romiplostim samples respectively.Conclusion The developed HPLC-CAD method for the determination of polysorbate 80in romiplostim has high sensitivity, specificity, repeatability and accuracy, which can be used for the determination of polysorbate 80 in the quality control of recombinant protein drugs.
