摘要
目的建立测定肺炎球菌多糖疫苗中苯酚含量的HPLC法。方法采用Novo-Pak C-18色谱柱（3.9 mm×150 mm,4μm）,以甲醇-水（20∶80）为流动相进行洗脱,流速为1.0 ml/min,检测波长为271 nm,柱温为25℃,进样量为10μl。对该方法的适用性、专属性、精密度、重现性、稳定性、准确性进行验证,并确定该方法的线性范围及定量限。用建立的方法检测6批23价肺炎球菌多糖疫苗中苯酚含量,并与溴量滴定法进行比较。结果苯酚浓度在0.031 25~0.500 2 mg/ml范围内,与峰面积呈良好的线性关系（r=1.000）,定量限为0.31μg/ml;供试品溶液的色谱峰与苯酚对照品溶液的主峰保留时间均约在4.5 min;苯酚对照品溶液连续进样6次,峰面积相对标准偏差（RSD）为0.09%,保留时间的RSD为0.02%;6份同1批供试品溶液中苯酚含量的均值为2.42 mg/ml,RSD为0.48%;同1份供试品溶液室温下放置,0~24 h测定12次,保留时间均值为4.485,RSD为0.53%,峰面积平均值为1 037 336,RSD为0.29%;9份加标供试品溶液的平均回收率为99.63%,RSD为1.18%。6批23价肺炎球菌多糖疫苗中苯酚含量的两种方法检测结果基本一致。结论建立的HPLC法操作简便,专属性、精密度、准确度、重现性、稳定性好,可对23价肺炎球菌多糖疫苗中的苯酚进行准确定量。
Objective To develop a high performance liquid chromatography（HPLC）method for determination of phenol content in pneumococcal polysaccharide vaccine. Methods The analysis were performed on a Novo-Pa K C-18 column（3. 9 mm × 150 mm,4 μm）. Methanol- ultrapure water（20 ∶ 80, v ∶ v）was used as a mobile phase at a flow rate of1. 0 ml / min. The detection wavelength was set at 271 nm, while the column temperature was 25 ℃, and the sample loading was 10 μl. The developed method was verified for suitability, specificity, precision, reproducibility, stability and accuracy, and determined for linear range and quantitative limit. The phenol contents in six batches of 23-valent pneumococcal polysaccharide vaccine were determined by the developed method, of which the results were compared with those by bromine content titration. Results The phenol concentration at a range of 0. 031 25 ~ 0. 500 2 mg / ml showed good linear relationship to the peak area（r = 1. 000）, while the quantitative limit was 0. 31 mg / ml. Both the retention times of chromatographic peak of test sample solution and the main peak of phenol control were about 4. 5 min. The phenol control was loaded for 6 times, of which the RSDs of peak area and retention time were 0. 09% and 0. 02%respectively. The mean phenol content in six samples of the same batch of test sample solutions was 2. 42 mg / ml, with a RSD of 0. 48%. The same test sample solution stored at room temperature was determined for 12 times within 24 h, of which the mean retention time and mean peak area were 4. 485 min and 1 037 336, with RSDs of 0. 53% and 0. 29%,respectively. The mean recovery rate of nine spike test sample solutions was 99. 63%, with a RSD of 1. 18%. The determination results of phenol contents in six batches of 23-valent pneumococcal polysaccharide vaccine were basically in agreement. Conclusion The developed HPLC method is simple, specific, precise, accurate, reproducible and stable,which may be used for accurate quantitation of phenol in 23-valent pn
出处
《中国生物制品学杂志》
CAS
CSCD
2015年第8期823-826,831,共5页
Chinese Journal of Biologicals
基金
国家高技术研究发展计划(863计划)(2012AA02A402)
关键词
高效液相色谱法
肺炎球菌多糖疫苗
苯酚
High performance liquid chromatography（HPLC）
Pneumococcal polysaccharide vaccine
Phenol
