Development and verification of a high-performance liquid chromatography method for determination of polysorbate 80 content in oil-in-water emulsions and its comparison with other methods
10.13200/j.cnki.cjb.004429
- VernacularTitle:水包油乳剂中聚山梨酯80含量高效液相色谱检测方法的建立、验证及与其他方法的比较
- Author:
JIA Lanxin
- Publication Type:Journal Article
- Keywords:
Oil-in-water emulsions;
Polysorbate 80;
High-performance liquid chromatography (HPLC);
Cobalt ammonium
- From:
Chinese Journal of Biologicals
2025;38(02):172-178+184
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop and verify a high-performance liquid chromatography(HPLC) method for the determination of polysorbate 80 in oil-in-water emulsions, and to compare it with the modified cobalt ammonium thiocyanate colorimetric method for the determination of polysorbate 80 in oil-in-water emulsions. Methods Eclipse Plus C8 column, TSKgel G2000SW_(XL) column and ChromCore SAA column were used to detect polysorbate 80 standard or test sample to determine the optimum chromatographic column, and the elution gradient of mobile phase B(50%, 75% and 90% as the elution gradient of mobile phase B in the second stage to detect polysorbate 80 standard solution; 90% and 75% as the elution gradient of mobile phase B in the second stage to detect test sample solution) and the flow rate(0. 8, 1. 0, 1. 2 mL/min) were optimized.The specificity, linear range, repeatability, accuracy and durability of the method were verified, and the linear range, repeatability and accuracy were compared with those of the modified cobalt ammonium thiocyanate colorimetric method. Results The conditions of HPLC method were as follows: ChromCore SAA chromatographic column was adopted at a column temperature of 25 ℃, the mobile phase A was aqueous solution containing 0. 1%(v/v) acetic acid, and the mobile phase B was isopropanol containing 0. 1%(v/v) acetic acid, with an injection volume of 10 μL and gradient elution with the optimum elution gradient of 75% for mobile phase B in the second stage at a flow rate of 1. 2 mL/min. Polysorbate 80 chromatographic peak was observed at 3. 1 min in both polysorbate 80 standard solution and test solution, and other chromatographic peaks were observed at 6 min in test solution. In the concentration range of 300-1 000 μg/mL, polysorbate 80 standard solution exhibited good linear relationship with the peak area, and the regression equation was: y = 1. 44 x + 4. 91, r = 0. 999 8. The RSD of polysorbate 80 content in six test solutions was 1. 13%. The spike recovery rates of low, medium and high content samples were 96. 51%-104. 82%. The RSD of polysorbate 80 content was 2. 38% after the test solution was placed at room temperature for 0, 2, 4, 6 and 8 hours. The linear relationship of the two methods was both good. The repeatability and accuracy of HPLC method were better than those of modified cobalt ammonium thiocyanate colorimetric method. Conclusion The developed HPLC method is superior to the modified cobalt ammonium thiocyanate colorimetric method with good specificity, precision, accuracy and durability, which provides a reliable method for the determination of polysorbate 80 in biological products containing various surfactants.
