A群脑膜炎球菌多糖原液中苯酚残留量气相色谱检测方法的建立及验证

Development and verification of gas chromatography for determination of residual phenol content in bulk of group A meningococcal polysaccharide

目的建立A群脑膜炎球菌多糖原液中苯酚残留量气相色谱检测方法,并进行验证。方法采用标准溶液加入法,以乙酸丁酯为萃取剂制备对照品及供试品溶液,采用气相色谱法进行检测,色谱条件为:Agilent DB-WAX[聚乙二醇(PEG)强极性柱30 m×0.25 mm×0.25μm]色谱柱,柱温165℃,进样口温度250℃,检测器温度300℃,流速2 m L/min,载气为氮气,进样分流比5∶1,进样量1μL。对方法的专属性及系统适用性、检测限和定量限、线性范围、精密度、准确度进行验证。采用建立的方法检测9批A群脑膜炎球菌多糖原液苯酚残留量。结果苯酚的保留时间分别为5.046 min,拖尾因子为1.04,与乙酸乙酯的分离度为33.21,理论塔板数大于5 000;检测限为0.535×10-3mg/m L,定量限为1.070×10-3 mg/m L;浓度在4~20μg/m L范围内,与峰面积呈良好的线性关系,相关系数(r)为0.999 9;同一操作人员连续重复检测6次结果的相对标准偏差(RSD)为0.4%,2名试验员不同时间重复检测6次结果的RSD为1.5%;9份加标供试品溶液的回收率为95.8%~100.0%;9批A群脑膜炎球菌多糖原液中苯酚残留量均符合规定。结论建立的A群脑膜炎球菌多糖原液中苯酚残留量气相色谱检测方法,具有良好的重复性及准确性,可用于A群脑膜炎球菌多糖原液中的苯酚残留量检测。

Objective To develop and verify a gas chromatography method for determination of residual phenol content in bulk of group A meningococcal polysaccharide. Methods Standard addition method was adopted. Control and sample solutions were prepared using butyl acetate as an extractant,and determined by gas chromatography. The chromatographic condition was as follows：a column of Agilent DB-WAX type（polyethylene glycol,strong polar column,30 m ×0. 25 mm × 0. 25 μm）was adopted,while the column temperature was 165 ℃,the inlet temperature was 250 ℃,and the detector temperature was 300 ℃. Nitrogen was selected as the carrier gas at a flow rate of 2 ml/min,while the sample split ratio was 5 ∶ 1 and sample volume was 1 μl. The method was verified for specificity,systemic suitability,detection limit,quantification limit,linear range,precision and accuracy,and used for determination of residual phenol content in9 batches of bulk of group A meningococcal polysaccharide. Results The retention time of phenol was 5. 046 min,while the tailing factor was 1. 04,the separation degree with ethyl acetate was 33. 21,the number of theoretical plates was more than 5 000. The detection and quantitative limits were 0. 535 × 10^-3 and 1. 070 × 10^-3 mg/m L respectively,while the phenol at a concentration of 4 to 20 μg/m L showed good linear relationship to the peak area,with a correlation coefficient of 0. 999 9. The RSD of six test results by one personnel was 0. 4%,while that by two personnel was 1. 5%.The spike recovery rates of nine samples were 98. 5% ~ 100%. All the residual phenol contents in nine batches of bulk of group A meningococcal polysaccharide met the relevant requirements. Conclusion The developed gas chromatograp hy showed high reproducibility and accuracy,which might be used for determination of residual phenol content in the bulk of group A meningococcal polysaccharide.