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目的:建立一种适用于新型抗凝血多肽Hirulog-S含量及杂质的分析方法,以及其水分和三氟乙酸(TFA)含量测定方法。方法:采用Zorbax Eclipse XDB-C18色谱柱(150 mm×4.6 mm,5μm),以含0.1%TFA的水为流动相A,含0.1%TFA的乙腈为流动相B,流速1.0 mL.min-1,检测波长214 nm,柱温25℃,测定样品中的含量及有关物质。采用卡尔菲休库仑滴定法测定水分,使用高效液相色谱法(HPLC)测其TFA含量。结果:在选定的色谱条件下,Hirulog-S与相邻杂质峰能较好分开,在25.4～508μg.mL-1范围内,峰面积与浓度线性关系良好。3批样品含量分别是99.7%±1.3%,100.4%±1.1%,99.4%±0.96%,总有关物质分别为0.59%～0.97%,样品中水分含量在2.1%～2.3%之间,TFA含量在9.08%～12.76%之间。结论:该方法快速简便,结果准确可靠,可用于Hirulog-S的质量分析。
OBJECTIVE: To establish an analytical method suitable for the determination of Hirulog-S content and impurities in new anticoagulant peptides, and to determine the content of water and trifluoroacetic acid (TFA). Methods: A mobile phase A was obtained on a Zorbax Eclipse XDB-C18 column (150 mm × 4.6 mm, 5 μm) using 0.1% TFA in water as mobile phase A and 0.1% TFA in acetonitrile as mobile phase B at a flow rate of 1.0 mL · min- , Detection wavelength 214 nm, column temperature 25 ℃, determination of the content of the sample and related substances. Water was determined by Coulometric coulometric titration and its TFA content was determined by high performance liquid chromatography (HPLC). Results: Under the selected chromatographic conditions, Hirulog-S can be separated from the adjacent impurity peaks well. The linear relationship between peak area and concentration is good in the range of 25.4 ~ 508μg.mL-1. The contents of three batches of samples were 99.7% ± 1.3%, 100.4% ± 1.1% and 99.4% ± 0.96% respectively, the total related substances were 0.59% ~ 0.97%, the contents of water in samples were between 2.1% and 2.3% Between 9.08% ~ 12.76%. Conclusion: The method is fast and simple, accurate and reliable, and can be used for the quality analysis of Hirulog-S.