Development and verification of a HPLC method for quantification of residual 2, 6-di-O-methyl-β-cyclodextrin content in DTacP intermediate products
10.13200/j.cnki.cjb.004740
- VernacularTitle:吸附无细胞百（三组分）白破联合疫苗中间产品中2,6-二-O-甲基-β环糊精残留量高效液相色谱检测方法的建立及验证
- Author:
Ling WANG
- Publication Type:Journal Article
- Keywords:
Diphtheria,tetanus and acellular pertussis combined vaccine,adsorbed(DTacP);
2,6-Di-O-methyl-β-cyclodextrin;
High-performance liquid chromatography(HPLC)
- From:
Chinese Journal of Biologicals
2026;39(07):837-845
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop and verify a high-performance liquid chromatography(HPLC) method for the determination of 2, 6-di-O-methyl-β-cyclodextrin residues in the intermediate products of diphtheria, tetanus and acellular pertussis combined vaccine, adsorbed(DTacP), so as to use it for the detection of 2, 6-di-O-methyl-β-cyclodextrin residues in purified and detoxified antigens of pertussis.Methods The chromatographic conditions were as follows: the chromatography column C18 was used, the mobile phase was 15% methanol aqueous solution with the flow rate of 0. 5 mL/min, the column temperature was set to 25 ℃, and the detection wavelength was 200 nm. The method was verified for specificity, linearity and range, accuracy, repeatability, intermediate precision, robustness, and limit of quantitation(LOQ). The established method was used to detect the 2, 6-di-O-methyl-β-cyclodextrin residues in two batches of intermediate products and finished products of DTacP.Results Residual 2, 6-di-O-methyl-β-cyclodextrin was detected at the target peak position in both purified antigen and detoxified antigen spiked samples, and the resolution of the target peaks was more than 2. The method exhibited good linearity in the range of 10 to 200 ??g/mL, r > 0. 99, with a LOQ of 10 ??g/mL. The RSDs of the test results of each spiked antigen in six samples were less than 5%, and the recovery rates were between 90% and 110%. The RSDs of the intra-assay precision of six samples were all less than 5%, and the RSDs of the inter-assay precision of the 12 samples prepared by different personnel were all less than 5%. When the flow rate and column temperature were adjusted by ± 20%,the recovery rates of the samples to be tested were between 90% and 110%. After purification, no residual 2, 6-di-O-methyl-β-cyclodextrin was detected in the detoxified antigens, bulk solutions and finished products.Conclusion An HPLC method for the determination of 2, 6-di-O-methyl-β-cyclodextrin residues in the intermediate products of DTacP was successfully established, which is simple to operate with high accuracy and good specificity, and suitable for detecting the 2, 6-di-O-methyl-β-cyclodextrin residues in the intermediate products of DTacP.
