四价流感病毒裂解疫苗中Triton X-100残留量高效液相色谱检测方法的建立、验证及与比浊法的对比

Development and verification of a method for determination of residual Triton X-100 content in quadrivalent influenza virus split vaccine by high performance liquid chromatography and comparison with turbidimetric method

目的建立四价流感病毒裂解疫苗中Triton X-100残留量高效液相色谱(high performance liquid chromatography,HPLC)检测方法,对方法进行验证,并与比浊法进行对比。方法对HPLC法的流动相比例(水∶甲醇=30∶70、20∶80、10∶90)、流速(1.0、1.2、1.4 mL/min)、进样量(5、10、20μL)进行筛选,确定HPLC法的色谱条件。对HPLC法和比浊法分别进行验证,并对两种方法进行对比。结果 HPLC法的色谱条件为:采用BDS HYPERSIL C18色谱柱(250 mm×4.6 mm,5μm);流动相:超纯水∶甲醇=20∶80;流速:1.2 mL/min;检测波长:230 nm;进样量:10μL;柱温:30℃。HPLC法中,Triton X-100色谱峰与杂质分离良好,在2~100μg/mL范围内线性关系良好,r为0.999 9;检测限为1μg/mL,定量限为2μg/mL,准确性和精密性良好。采用比浊法检测时,标准品在10~100μg/mL范围内线性关系良好,r为0.996 5,定量限为10μg/mL。结论与比浊法相比,HPLC法更适用于四价流感病毒裂解疫苗中Triton X-100残留量的测定。

Objective To develop and verify a high performance liquid chromatography(HPLC)method for determination of residual Triton X-100 content in quadrivalent influenza split vaccine, and compare with turbidimetric method.Methods The condition for HPLC was determined based on the optimization of ratio of water to methanol in mobile phase(30 ∶ 70,20 ∶ 80 and 10 ∶ 90),flow rate(1. 0,1. 2 and 1. 4 mL/min)and sample loading(5,10 and 20 μL). The developed HPLC method and turbidimetric method were verified separately and compared. Results The condition of HPLC was as follows:BDS HYPERSIL C18 column(250 mm × 4. 6 mm,5 μm) was adopted,serving the mixture of water and methanol(20 ∶ 80)as mobile plase at a flow rate of 1. 2 mL/min. The detection wavelength,sample loading and column temperature were 230 nm,10 μL and 30 ℃ respectively. The Triton X-100 and the impurities were separately well by HPLC,of which the linear range of curve was 2 ~ 100 μg/mL(r = 0. 999 9). The limits of detection and quantitation of the developed method were 1 and 2 μg/mL respectively,indicating high accuracy and precision.However,the linear range of turbidimetric method for determination of standard was 10 ~ 100 μg/mL(r = 0. 996 5),while the limit of quantitation was 10 μg/mL. Conclusion Compared with turbidimetric method,HPLC is more suitable for the determination of residual Triton X-100 content in quadrivalent influenza virus split vaccine.