1型肺炎球菌荚膜多糖中十六烷基三甲基溴化铵含量反相高效液相色谱检测方法的建立及验证  

Development and validation of reversed-phase high performance liquid chromatography for determination of residual cetyltrimethylammonium bromide content in serotype 1 pneumococcal polysaccharide

作　　者：陈思 邓前 曾华英 邹莎莎 张伟 但傲欢 廖红梅 赵静 刘威 CHEN Si;DENG Qian;ZENG Hua-ying;ZOU Sha-sha;ZHANG Wei;DAN Ao-huan;LIAO Hong-mei;ZHAO Jing;LIU Wei(Chengdu Institute of Biological Products,Chengdu 610023,Sichuan Province,China)

机构地区：[1]成都生物制品研究所,成都四川610023

出　　处：《中国生物制品学杂志》2020年第10期1192-1195,1200,共5页Chinese Journal of Biologicals

摘　　要：目的建立检测1型肺炎球菌荚膜多糖(简称Pn1型多糖)中残留十六烷基三甲基溴化铵(cetyltrimethylammonium bromide,CTAB)含量的反相高效液相色谱(reversed-phasehigh performance liquid chromatography,RP-HPLC)方法,并进行验证。方法色谱条件:选用反相色谱柱Waters/XSelect CSH C18(4.6 mm×250 mm,5μm),流动相为含三氟乙酸(trifluoroacetic acid,TFA)的10%甲醇水溶液,梯度洗脱,流速为1.0 mL/min,柱温为20℃,检测波长为206 nm。优化流动相甲醇中TFA的比例(0.01%、0.05%和0.1%)。Pn1型多糖用100μg/mL脱氧胆酸钠(sodium deoxycholate,DOC)水溶液溶解,超滤离心后上样。验证方法的专属性、线性范围、灵敏度、准确性及重复性。应用该方法检测3批Pn1型多糖样品CTAB残留量。结果选择含0.01%TFA的10%甲醇水溶液作为流动相;加标样品除有CTAB对照品色谱峰外,还有2个杂质峰,且3个峰均达到基线分离,表明方法专属性良好;CTAB对照品在0.2~4.0μg/m L浓度范围内与峰面积线性良好,r2>0.999;检出限及定量限分别为0.1和0.2μg/mL;0.2、0.4及0.6μg/mL浓度的加标样品回收率分别为101.18%、100.85%和101.72%,平行检测3次的RSD分别为1.69%、1.98%和0.76%。3批样品CTAB残留量均<0.324μg/mL,符合内控要求。结论成功建立了检测Pn1型多糖中残留CTAB含量的RP-HPLC法,该方法具有良好的专属性、线性、准确性,灵敏度高,重复性好,可用于肺炎球菌荚膜多糖中的残留CTAB检测。Objective To develop and validate a reversed-phase high performance liquid chromatography(RP-HPLC)for determination of residual cetyltrimethylammonium bromide(CTAB)content in serotype 1 pneumococcal polysaccharide(Pn1).Methods Reversed-phase Waters/XSelect CSH C18(4.6 mm×250 mm,5μm)chromatographic column was adopted serving 10%methanol aqueous solution containing trifluoroacetic acid(TFA)as a mobile phase by gradient elution at a flow rate of 1.0 mL/min,a column temperature of 20℃and a detection wavelength of 206 nm.The proportion(0.01%,0.05%and 0.1%)of TFA in methanol aqueous solution as mobile phase was optimized.The Pn1 samples were dissolved in 100μg/mL sodium deoxycholate(DOC)aqueous solution then ultrafiltrated,centrifuged and loaded.The developed method was validated for specificity,linear range,sensitivity,accuracy and repeatabicity.The residual CTAB contents in three batches of Pn1 were determined by the method.Results The optimal mobile phase was10%methanol aqueous solution containing 0.01%TFA.In addition to the chromatographic peak of CTAB control,two peaks of foreign matters were observed on the chromatographic profile of spike samples,all of which were baseline separated,indicating good specificity of the developed method.The linear range of the method for determination of CTAB reference was 0.2~4.0μg/mL,with a r^2 value of more than 0.999.The limit of detection(LOD)was 0.1μg/m L,while the limit of quantification(LOQ)was 0.2μg/mL.The spike recovery rates of CTAB at concentrations of 0.2,0.4 and 0.6μg/mL were 101.18%,100.85%and 101.72%,while the RSDs of determination results in three tests were 1.69%,1.98%and 0.76%,respectively.All the residual CTAB contents in three batches of CTAB were less than0.324μg/m L,which met the requirements of internal control.Conclusion The RP-HPLC method for determination of residual CTAB content in Pn1 was successfully developed,which showed good specificity,linearity,accuracy,sensitivity and repeatability.

关 键 词：1型肺炎球菌荚膜多糖 十六烷基三甲基溴化铵 反相高效液相色谱 脱氧胆酸钠 

分 类 号：R392-33[医药卫生—免疫学]