Development and verification of a reversed-phase high-performance liquid chromatography method for the determination of biotin residues in human papillomavirus vaccines
10.13200/j.cnki.cjb.004782
- VernacularTitle:人乳头瘤病毒疫苗生物素残留量反相高效液相色谱检测方法的建立及验证
- Author:
Xiaofeng LIU
- Publication Type:Journal Article
- Keywords:
Quadrivalent human papillomavirus(HPV) vaccine;
Biotin;
Reversed-phase high-performance liquid chromatography(RP-HPLC)
- From:
Chinese Journal of Biologicals
2026;39(10):1205-1212
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop and verify a reversed-phase high-performance liquid chromatography(RP-HPLC) method for quantifying biotin residues in human papillomavirus(HPV) vaccines, thereby providing data support for the research on the removal ability of biotin in the downstream production process of HPV vaccines.Methods Chromatographic parameters were systematically optimized, including mobile phase composition, elution gradient, and temperature controls. A RP-HPLC method for the determination of biotin residues in HPV vaccines was developed, and was verified for system suitability, specificity, linearity, accuracy, precision, limit of detection(LOD), limit of quantitation(LOQ), and robustness in strict accordance with the Guideline for Analytical Method Validation(General Chapter 9101) of the Chinese Pharmacopoeia(Vol Ⅲ, 2025edition). The validated method was subsequently applied to detect biotin residues in the in-process samples and bulk solution samples of quadrivalent HPV vaccines.Results The optimized chromatographic conditions were as follows: the column was Luna C18~??100??(4. 6 mm × 250 mm, 5 ??m); the mobile phase A, B and C were water, acetonitrile and 0. 1% trifluoroacetic acid(TFA) respectively with ternary gradient; the flow rate was 1 mL/min, with detection at 200 nm, column temperature at35 ℃, autosampler temperature at 25 ℃, and an injection volume of 100 ??L. The method exhibited good system applicability and specificity. The biotin showed a good linear relationship in the concentration range of 0. 312 5-5 ??g/mL(R~2= 1).The average recovery rate of high-, medium-, and low-concentration(4, 1. 5, 0. 5 ??g/mL) biotin quality control(QC) samples was 100. 28%. The relative standard deviations(RSDs) of repeatability test results for low-concentration biotin QC samples were ≤ 5%. The RSDs of intermediate precision test results for low-concentration biotin QC samples prepared over three days were ≤ 5%, the LOQ was 0. 312 5 ??g/mL, and the LOD was 0. 156 ??g/mL. The RSDs of the test results of low-concentration biotin QC samples stored at 25 ℃ for 0, 24 and 48 h were ≤ 5%, indicating good stability. No biotin residues were detected in either in-process samples or bulk solution samples of three batches of quadrivalent HPV vaccines by using this method.Conclusion A RP-HPLC method for the determination of biotin residues was established. This method exhibits excellent performance and is suitable for the investigation of biotin residues in quadrivalent HPV vaccines.
