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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 establish a HPLC method for the determination of phenol in pneumococcal polysaccharide vaccine. Methods The Novo-Pak C-18 column (3.9 mm × 150 mm, 4 μm) was used. The mobile phase consisted of methanol and water (20:80) with a flow rate of 1.0 ml / min and a detection wavelength of 271 nm. The temperature was 25 ℃, the injection volume was 10μl. The applicability, specificity, precision, reproducibility, stability and accuracy of the method were verified, and the linear range and the limit of quantitation of the method were determined. The phenol content in 6 batches of 23-valent pneumococcal polysaccharide vaccine was detected by the established method and compared with the bromine titration method. Results The phenol concentration in the range of 0.031 25-0.500 2 mg / ml showed a good linear relationship with the peak area (r = 1.000) and the limit of quantification was 0.31 μg / ml. The chromatographic peak of the test solution was similar to that of the phenol control solution The main peak retention time was about 4.5 min; Phenol reference solution 6 consecutive injections, peak area relative standard deviation (RSD) was 0.09%, RSD retention time of 0.02%; 6 with the same batch of test solution of phenol With a mean of 2.42 mg / ml and a RSD of 0.48%. The same solution was stored at room temperature for 12 times at 0-24 h with a mean retention time of 4.485 and a RSD of 0.53% with an average peak area of 1 037 336 and RSD 0.29%. The average recovery of 9 spiked samples was 99.63% and the RSD was 1.18%. The test results of the two methods of phenol content in six batches of 23-valent pneumococcal polysaccharide vaccine were basically the same. Conclusion The established HPLC method is simple, specific, accurate, accurate, reproducible and stable, and can accurately quantify phenol in 23-valent pneumococcal polysaccharide vaccine.