Abstract: Objective To evaluate the applicability of a high-performance liquid chromatography-fluorescence micelle assay (HPLC-FMA) for quantitative determination of polysorbate 80 in human papillomavirus (HPV) vaccines. Methods A SupelcoKnitted Reactor Coil (5 m×0.50 mm ID) was used as the reaction coil for HPLC-FMA analysis. The mobile phase was a tris (hydroxymethyl) aminomethane mixed solution at a flow rate of 1.5 mL·min^-1. The detection wavelength was set at an excitation of 350 nm and an emission of 420 nm while the acquisition time was 2.5 minutes. The contents of polysorbate 80 were calculated using the fluorescence intensity (peak area). Results The method proved to be highly specific. Polysorbate 80 showed a good linear relationship at concentrations ranging from of 50 to 250 µg·mL^-1, with an R² value of 0.999 8. The instrument precision of RSD was 0.223%, and the intermediate precision of RSD was 1.269%. The average value from six replicate measurements was 91.123 µg·mL^-1 with an RSD of 0.399%. Recovery rates at different concentrations ranged from 98.725% to 99.866%, with RSD values between 0.321% to 1.636%. The contents of polysorbate 80 in eight batches were determined as 96.500, 95.850, 94.940, 96.049, 91.847, 92.147, 102.060 and 84.667µg·mL^-1, respectively. Conclusion This HPLC-FMA method requires no toxic chemicals or chromatographic columns, and can complete the assay within minutes, which can facilitate rapid, accurate, and reliable quantification of polysorbate 80 in HPV vaccines.

Key words: HPV Vaccine, Human Papillomavirus(HPV), Polysorbate 80 Content, Fluorescence Micelle Assay, High Performance Liquid Chromatography, N-Phenyl-1-Naphthylamine, Pharmacopoeia