多糖疫苗中十六烷基三甲基溴化铵和去氧胆酸残留量液相色谱串联质谱检测方法的建立及验证

Development and verification of liquid chromatography tandem mass spectrometry for simultaneous determination of residual hexadecyltrimethyl ammonium bromide and sodium deoxycholate contents in polysaccharide vaccines

目的建立多糖疫苗中十六烷基三甲基溴化铵(hexadecyltrimethyl ammonium bromide,CTAB)和去氧胆酸(deoxycholic acid,OBA)残留量的液相色谱串联质谱(liquid chromatography tandem mass spectrometry,LC-MS/MS)检测方法,并进行验证。方法建立以Kinetex色谱柱C18(2.1 mm×50 mm,2.6μm)为固定相,5 mmol/L甲酸铵、乙腈为流动相的LC-MS/MS法,并验证方法的线性范围、精密性、准确度、检出限及定量限。采用建立的方法检测23批肺炎球菌多糖(1、2、3、4、5、6B、7F、8、9N、9V、10A、11A、12F、14、15B、17A、18C、19A、19F、20、22F、23F和33F)、3批b型流感嗜血杆菌(Haemophilus influenzae type b,Hib)粗糖和精糖、3批A、C、Y、W135群脑膜炎球菌粗糖和精糖中CTAB及OBA残留量。结果 CTAB浓度在1.56~50μg/L范围时,与峰面积呈良好的线性关系,回归方程为Y=133 056 X-290 15,R2=0.999 9;OBA浓度在6.25~100μg/L范围时,与峰面积呈良好的线性关系,回归方程为Y=250.5 X-59.1,R2=0.999 7;OBA及CTAB对照品6次重复进样检测的保留时间和峰面积RSD均<2%;OBA和CTAB加标回收率均在91.8%~103.5%之间;OBA的检出限为1.02μg/L,定量限为2.04μg/L,CTAB的定量限为1.56μg/L。4、9N、9V和15B型肺炎球菌多糖无明显CTAB检出,其他血清型均有不同程度检出;大部分血清型肺炎球菌多糖中无明显OBA检出。3批A、C、Y、W135脑膜炎球菌粗糖中CTAB残留量为112.40~1 709.60μg/L,对应批次精糖中CTAB残留量为0.08~43.20μg/L。3批Hib粗糖中CTAB残留量分别为1 265.10、1 189.00和1 513.30μg/L,对应批次精糖中CTAB残留量分别为6.50、166.60、6.65μg/L。结论建立了多糖疫苗中CTAB和OBA的LC-MS/MS检测方法,该方法具有良好的精密性及准确度,可用于多糖疫苗及多糖结合疫苗中CTAB和OBA残留量的检测。

Objective To develop and verify a liquid chromatography tandem mass spectrometry(LC-MS/MS)method for simultaneous determination of residual hexadecyltrimethyl ammonium bromide(CTAB)and deoxycholic acid(OBA)in polysaccharide vaccines.Methods A LC-MS/MS method was developed by using KinetexC18(2.1 mm × 50 mm,2.6 μm)as stationary phase and 5 mmol/L ammonium for mate-acetonifrile as mobile phase,and verified for linear range,precision,accuracy,limit of detection(LOD)and limit of quantitation(LOQ).Twenty-three batches of pneumococcal polysaccharide(serotypes 1,2,3,4,5,6 B,7 F,8,9 N,9 V,10 A,11 A,12 F,14,15 B,17 A,18 C,19 A,19 F,20,22 F,23 F and33 F),three batches of crude and the corresponding purified Haemophilus influenzae type b(Hib)polysaccharide and three batches of crude and the corresponding purified groups A,C,Y and W135 meningococcal polysaccharide were determined for residual CTAB and OBA contents by the developed method.Results The CTAB at concentrations of 1.56~50 μg/L showed good linear relationship to the peak area,of which the regression equation was Y = 133 056 X-290 15(R2= 0.999 9).However,the OBA at concentrations of 6.25~100 μg/L showed good relationship to the peak area,of which the regression equation was Y = 250.5 X-59.1(R2= 0.999 7).All the RSDs of retention time and peak area of OBA and CTAB in six repeat tests were less than 2%.The spike recovery rates of OBA and CTAB were 91.8%~103.5%.The LOD and LOQ of OBA were 1.02 and 2.04 μg/L respectively,while the LOQ of CTAB was 1.56 μg/L.CTAB was undetected in pneumococcal polysaccharide of serotypes 4,9 N,9 V and 15 B,while was detected at different levels in other serotypes.However,no OBA was detected in most of the serotypes.The residual CTAB contents in three batches of crude groups A,C,Y and W135 meningococcal polysaccharide were 112.40~1 709.60 μg/L,while those in the corresponding purified ones were 0.08 ~43.20 μg/L.However,the residual CTAB contents in three batches of crude Hib polysaccharide were 1 265.10,1 189.00 and 1 513.30 μg/L,while those in the corresponding purified polysaccharide were 6.50,166.60 and 6.65 μg/L,respectively.Conclusion A LC-MS/MS method was developed,which was precise and accurate,and might be used for deter-mination of residual CTAB and OBA contents in polysaccharide vaccines and polysaccharide conjugate vaccines.