Establishment and verification of a method for determination of aluminum content in vaccines by high performance liquid chromatography-diode array detection with 8-hydroxyquinoline pre-column derivatization
10.13200/j.cnki.cjb.004616
- VernacularTitle:疫苗中铝含量8-羟基喹啉柱前衍生-高效液相色谱-二极管阵列检测方法的建立及验证
- Author:
Yijie LIU
1
Author Information
1. Fujian Institute for Food and Drug Quality Control, Fuzhou 350005, Fujian Province, China
- Publication Type:Journal Article
- Keywords:
High performance liquid chromatography-diode array detection(HPLC-DAD);
Vaccines;
Aluminum content;
8-Hydroxyquinoline
- From:
Chinese Journal of Biologicals
2025;38(12):1490-1497
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish an 8-hydroxyquinoline pre-column derivatization-high performance liquid chromatographydiode array detection(HPLC-DAD) method for the determination of aluminum content in vaccines, and to verify the method for vaccine quality control.Methods The determination was carried out by pre-column derivatization with 8-hydroxyquinoline, and the sample pretreatment conditions of hydrochloric acid solution concentration(0. 1, 0. 5, 1, 2, 5 mol/L),hydrochloric acid digestion time(20, 30, 40, 50, 60 min), concentratio%n of derivatization reagent 8-hydroxyquinoline(0. 5,1, 2, 3, 4 mg/mL), and ammonia concentration(1. 25%, 6. 25%, 12. 50%, 18. 75%, 25%) were optimized. ZORBAX SB-C_8(4. 6 mm × 150 mm, 5 ??m) column was used with methanol-water as mobile phase in gradient elution at a flow rate of0. 8 mL/min. The detection wavelength was 254 nm, the column temperature was 30 ℃, and the injection volume was 10 ??L.The linear range, accuracy, precision, specificity and stability of the method were validated, the limit of detection(LOD) and limit of quantification(LOQ) were determined, and the method was used to detect the aluminum content in 16 batches of bivalent human papillomavirus(HPV) and three batches of recombinant hepatitis E virus(HEV) vaccines.Results The optimized pretreatment conditions were as follows: hydrochloric acid concentration 1 mol/L, digestion time 20 min,derivatization reagent 8-hydroxyquinoline concentration 2 mg/mL, and ammonia concentration 12. 5%. Aluminum showed a good linearity in the concentration range of 2 to 100 ??g/mL, with a correlation coefficient(r) of 0. 999 98. The average recovery rates of spiked samples were between 89. 6% and 95. 7%. The relative standard deviation(RSD) of the aluminum content test results of six samples was 1. 4%. The blank reagent showed no interference with the detection. The sample solution exhibited good stability at room temperature for 12 h. The LOD and LOQ of the method were 0. 267 ??g/mL and0. 820 ??g/mL respectively. The established method was used to detect the aluminum content in 16 batches of HPV vaccines and three batches of recombinant HEV vaccines, and the results were not significantly different from those detected by titration method in General Principles 3106 of the Chinese Pharmacopoeia(Volume Ⅲ, 2020 edition).Conclusion The established HPLC-DAD method is accurate and reliable, with good precision, specificity and stability. The mobile phase is only methanol-water without adding buffer salts and derivatizing agents, etc., and it is easy to operate and friendly to instruments and chromatographic columns. The method requires a small amount of samples, which can realize the detection of a single dose of vaccine sample and provide reference for the quality control of vaccines.
