目的 分析不同厂家珍珠明目滴眼液及珍珠液中蛋白多肽类成分的差异,为保证原料药生产工艺的稳定性,有效地控制珍珠明目滴眼液的质量提供参考依据。方法 采用UPLC-Q-TOF MS 正离子模式进行分析,鉴定珍珠明目滴眼液及珍珠液中的肽类成分,Acquity UPLC BEH C₁₈(50 mm×2.1 mm,1.7 μm)色谱柱,乙腈-0.1%甲醇水溶液作为流动相;柱前衍生化HPLC-DAD 法测定珍珠明目滴眼液中16 种氨基酸。结果 不同生产企业的珍珠明目滴眼液及珍珠液中多肽的相对分子质量(M_W)、16 种氨基酸分别占总氨基酸量的百分比差异较大。5 家企业中3 家的原料药珍珠液中肽的M_W 均小于2 000,另2 个厂家珍珠液中肽的M_W 为500~4 746,以大于2 000 的肽为主。7 个厂家的珍珠明目滴眼液样品中,2 个厂家样品中肽的M_W 以2 000~4 000 为主,并含有4 000 以上的肽,另5 个厂家样品中不含4 000 以上的肽。7 个厂家的珍珠明目滴眼液中16 种氨基酸质量浓度相对总氨基酸质量浓度的百分比差异较大,2 个厂家采用酸/酶水解工艺的珍珠液中脯氨酸与丙氨酸质量浓度的比值 > 1,5 个厂家采用水直接提取工艺的二者比值小于1。结论 建议修订珍珠液质量标准,明确制备工艺为水直接提取,确保滴眼液中的有效成分多肽(M_W 在500~5 000),珍珠液及珍珠明目滴眼液质量标准增加检查项脯氨酸与丙氨酸质量浓度的比值及定量测定项氨基酸总量,以有效控制珍珠明目滴眼液的质量,确保其有效性。

Objective Analysis for the differences of protein polypeptide ingredients in Zhenzhu Mingmu Eye Drops and Zhenzhuye in order to provide the reference data to ensure the stability of production process of crude material and effectively control the quality of Zhenzhu Mingmu Eye Drops. Methods Polypeptide ingredients in Zhenzhu Mingmu Eye Drops and Zhenzhuye were identified by UPLC-Q-TOF MS in positive mode using an Acquity UPLC BEH C₁₈ (50 mm×2.1 m m, 1.7 μ m) c olumn. M obile p hase i s acetonitrile-0.1% methanol; Contents of 16 amino acids were detected by pre-column derivatization RP-HPLC method. Results Molecular weight (M_W) of polypeptide and the contents of the amino acids in Zhenzhu Mingmu Eye Drops and Zhenzhuye were significantly different. In the five manufacturers, M_W of polypeptide was less than 2 000 in Zhenzhuye samples from the three manufacturers, while M_W of polypeptides were 500-4 746 for samples of the other two manufacturers and M_W of main polypeptides were higher than 2 000. M_W ranges of main polypeptides in Zhenzhu Mingmu Eye Drops from two manufacturers were 2 000-4 000 and beyond 4 000. There were not the polypeptides with M_W beyond 4 000 in samples of the other five manufacturers. The content percentages of 16 amino acids in Zhenzhu Mingmu Eye Drops from the seven manufacturers were obviously different. The mass concentration ratios of proline and alanine in Zhenzhuye produced by acid/enzyme hydrolysis from two manufacturers are beyond 1. For the other five manufacturers with water extract technique, the mass concentration ratios of proline and alanine are below 1. Conclusion The quality standard of Zhenzhuye should be revised and water extract should be applied in preparation process to ensure the effective ingredients polypeptides (500-5 000). In quality standard of Zhenzhu Mingmu Eye Drops and Zhenzhuye, the mass concentration ratio of proline/alanine and the total contents of amino acids should be added as check and content determination items, respectively, so as to effectively control the quality of Zhenzhu Mingmu Eye Drops and ensure its validity.