目的: 通过对新型填料色谱柱及流动相的筛选,建立高效液相色谱法检测氨甲环酸注射液中的有关物质,避免现行方法中离子对试剂的使用并降低盐的浓度。方法: 采用Thermo Mixed-mode WCX色谱柱(150 mm×4.6 mm,5 μm),以10 mmol·L-1磷酸二氢钠溶液(用氢氧化钠溶液调pH至5.2±0.05)-水-乙腈(50∶5∶45)为流动相,等度洗脱,流速1.0 mL·min-1,柱温25 ℃,检测波长210 nm,进样量20 μL。结果: 氨甲环酸与4个已知杂质的色谱峰均能良好分离。氨甲环酸、杂质B、杂质C、杂质D和杂质E在一定浓度范围内与相应的峰面积呈良好的线性关系(r≥0.999);检测限分别为0.34、0.50、0.005 6、0.002 1、0.12 μg·mL-1;4个已知杂质的平均加样回收率(n=9)分别为97.4%、100.5%、98.4%和96.6%,RSD分别为3.9%、0.24%、0.52%和1.4%;供试品溶液和对照品溶液在22 h内均稳定。5批样品中其他单杂检出个数及杂质总量均优于2020年版《中华人民共和国药典》方法。结论: 本方法专属性强,灵敏度高,仅添加低浓度盐即实现氨甲环酸与4个已知杂质的良好分离,适用于氨甲环酸注射液中有关物质的测定。
Objective: To establish a new HPLC method for the determination of the related substances in tranexamic acid injection by screening new type of HPLC columns, which can avoid the use of ion pair reagents in current methods and reduce salt concentration. Methods: The method was carried out using a Thermo Mixed-mode WCX column (150 mm×4.6 mm, 5 μm), the mobile phase consisted of 10 mmol·L-1 sodium dihydrogen phosphate solution (adjust the pH value to 5.2±0.05 with sodium hydroxide solution)-water-acetonitrile (50∶5∶45), the flow rate was 1.0 mL·min-1, the column temperature was 25 ℃, the detection wavelength was 210 nm and the injection volume was 20 μL. Results: The chromatographic peaks of tranexamic acid and its four impurities B, C, D and E were all separated. Good linear relationship was shown between the concentration of all the five compounds and their corresponding peak areas (r≥0.999). The LODs were 0.34, 0.50, 0.005 6, 0.002 1, 0.12 μg·mL-1. The average recoveries (n=9) were 97.4%, 100.5%, 98.4% and 96.6% with RSDs of 3.9%, 0.24%, 0.52% and 1.4%, respectively. The test solution and standard solution were all stable within 22 h. The detection results of 5 batches of tranexamic acid injection showed that the number of unspecified impurities and the total impurities content by using the new method were better than the current ChP method. Conclusion: The established method is specified and sensitive. Good separation can be achieved with low concentration of phosphate. Its applicable to the determination of related substances in tranexamic acid injection.
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