Development and verification of HPLC for determination of residual EDTA disodium salt in freeze-dried rabies vaccine(Vero cells) for human use
10.13200/j.cnki.cjb.004542
- VernacularTitle:冻干人用狂犬病疫苗（Vero细胞）原液中乙二胺四乙酸二钠残留量高效液相色谱检测方法的建立及验证
- Author:
FENG Ziyi
- Publication Type:Journal Article
- Keywords:
Freeze-dried rabies vaccine(Vero cells) for human use;
High-performance liquid chromatography(HPLC);
EDTA disodium salt(EDTA-2Na);
Residual content
- From:
Chinese Journal of Biologicals
2025;38(08):962-968
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop a high-performance liquid chromatography(HPLC) method for the determination of residual EDTA disodium salt(EDTA-2Na) content in freeze-dried rabies vaccine(Vero cells) for human use, and to verify and preliminarily apply the method, so as to ensure the content of EDTA-2Na within the safe concentration range.Methods The condition for HPLC was as follows: reverse chromatographic column was adopted, using the mobile phase consisting of 0. 1%phosphate solution as the aqueous phase and 100% methanol as the organic phase at a flow rate of 1. 0 mL/min and a detection wavelength of 242 nm. The injection volume was 20 μL, and the column temperature was 25 ℃. The developed method was verified for suitability, limit of quantitation(LOQ), specificity, linear range, accuracy, reproducibility, and intermediate precision, and the residual EDTA-2Na contents in thr ee batches of bulk of freeze-dried rabies vaccine(Vero cells) for human use were determined using this method.Results The LOQ of EDTA-2Na detected by this method was 0. 490 0 μg/mL. The blank and sample solution had no interference to EDTA-2Na, and the separation degree of the target and adjacent peaks was more than 1. 5. EDTA-2Na showed good linearity in the concentration range of 0. 490 0 to 9. 800 0 μg/mL. The recovery rates of EDTA-2Na in spiked samples at concentrations of 50%, 100% and 150% were from 94% to 113%. The recovery rates of EDTA-2Na in six solutions of reproducibility verification ranged from 100% to 102%, with a relative standard deviation(RSD) of 1%. A total of 12 solutions of intermediate precision verification from different personnel were measured, and the RSD of recovery rate was 2%. In addition, EDTA-2Na was not detected in three batches of samples.Conclusion The developed HPLC has good suitability, specificity, linearity, accuracy, reproducibility and intermediate precision, which is suitable for the determination of residual EDTA-2Na in bulk of freeze-dried rabies vaccine(Vero cells) for human use.
