目的: 建立超高效液相色谱法测定重组人生长激素原液及注射液中的有关物质。方法:采用Waters BEH C18 300Å色谱柱(150 mm×2.1 mm,1.7 μm),以12.5 mmol·L-1磷酸氢二钾缓冲溶液-乙腈为流动相,梯度洗脱,流速0.3 mL·min-1,检测波长220 nm,柱温45 ℃。结果:在选定的色谱条件下,能分离出较多有关物质,且主峰与各有关物质峰均能良好分离;质量浓度在0.25~5 mg·mL-1的范围内线性关系良好。精密度、耐用性及稳定性(72 h内)良好。根据质谱定性结果,探索了制备脱酰胺杂质、氧化杂质、脱水杂质等主要有关物质的处理方法。对于重组人生长激素注射液,本方法与现有标准相比,能够弥补我国注册标准方法中检测有关物质数量少,彼此间分离度不高的缺点,同时也克服了欧洲药典需要2种机理不同的独立实验方能完成有关物质控制的问题。结论:本方法适用于测定重组人生长激素原液及注射液中的有关物质。
陆俊杰, 李晶, 陈莹, 李懿, 张伟, 张慧, 吕萍,秦希月, 高向东, 梁成罡
. 重组人生长激素原液及注射液中有关物质UPLC分析方法建立*[J]. 药物分析杂志, 2022
, 42(1)
: 41
-50
.
DOI: 10.16155/j.0254-1793.2022.01.05
Objective: To establish a UPLC method for determination of related substances in recombinant human growth hormone bulk and injection. Methods: The analysis was conducted on Waters BEH C18 300Å column (150 mm×2.1 mm, 1.7 μm). The mobile phases were 12.5 mmol·L-1 dipotassium hydrogen phosphate buffer solution and acetonitrile with gradient elution, the flow rate was 0.3 mL·min-1, the column temperature was 45 ℃, and the detection wavelength was 220 nm. Results: Under the selected chromatographic conditions, more related substances could be separated, and the main peak and each peaks of related substances could be well separated. The linear relationship was good in the concentration range of 0.25-5 mg·mL-1. Precision, and durability was good. Stability within 72 h was good. According to the qualitative results of mass spectrometry. The method was explored that preparing the main related substances such as deamination impurities, oxidation impurities and dehydration impurities. For recombinant human growth hormone injection,compared with the existing standard, this method could compensate for the shortcomings of the registration standard such as the small number of substances and the low degree of separation between each other. and overcome the problem thatthe European Pharmacopoeia requires two independent experiments with different mechanisms to complete the control of related substances. Conclusion: This method is suitable for the determination of related substances in recombinant human growth hormone bulk and injection.
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