Establishment and validation of a pre-column derivatization-high performance liquid chromatography method for determination of free formaldehyde content in Sabin strain inactivated poliovirus vaccine(Vero cells)
10.13200/j.cnki.cjb.004402
- VernacularTitle:Sabin株脊髓灰质炎灭活疫苗（Vero细胞）中游离甲醛含量柱前衍生-高效液相色谱检测方法的建立及验证
- Author:
YANG Huan
- Publication Type:Journal Article
- Keywords:
High performance liquid chromatography(HPLC);
Inactivated poliovirus vaccine(IPV);
Formaldehyde;
Content determination
- From:
Chinese Journal of Biologicals
2025;38(1):53-60
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish a pre-column derivatization-high performance liquid chromatography(HPLC) method for the determination of free formaldehyde in Sabin strain inactivated poliovirus vaccine(IPV)(Vero cells), validate and apply the method, so as to provide a new method for the determination of free formaldehyde in vaccines.Methods The sample was derivatized with 2, 4-dinitrophenylhydrazine and loaded onto a C18 chromatographic column(5 μm, 120 ?, 4. 6 mm ×250 mm). The detection wavelength, mobile phase ratio, flow rate, derivatization time, temperature, buffer solution, and derivatization container were optimized for the separation conditions. The specificity, linearity, repeatability, accuracy, limit of detection(LOD), limit of quantitation(LOQ) and robustness of the method were verified. The content of free formaldehyde in 20 batches of IPV(Vero cells) was detected by using the optimized method.Results Chromatographic conditions: acetonitrile and water in a 70∶30 volume ratio as mobile phase, flow rate 0. 6 mL/min, determination at a wavelength of 352 nm.Derivatization conditions: 0. 5 mL of acetonitrile solution containing 2, 4-dinitrophenylhydrazine and 0. 25 mL of pH 5. 0buffer were added, followed by a 20 min incubation in 60 ℃ water bath. This chromatographic separation conditions effectively separated 2, 4-dinitrophenylhydrazine and formaldehyde derivatives, and the acetaldehyde had no effect on the determination results. In the range of 0. 05-100 μg/mL, formaldehyde standard concentration exhibited a good linear relationship with the peak area, with the r value of 0. 999 9. The relative standard deviations(RSDs) of six test results in the repeatability test was 0. 36%. The recovery rates of formaldehyde content in nine samples were between 102. 0% and 107. 0%. The LOD and LOQ were 0. 025 and 0. 05 μg/mL, respectively. The sample remained stable for 48 h after derivation, showing good robustness. The results of the same batch of samples had good repeatability, and the formaldehyde content was between 4. 5-9. 9 μg/dose.Conclusion The established method has the advantages of wide measurement range, good linearity and high accuracy, and can accurately quantify free formaldehyde in Sabin strain IPV(Vero cells), which can be used as an auxiliary detection method for free formaldehyde content in vaccine products, and is of great significance to the lot release and quality supervision for vaccines.
