HPLC法测定坎格雷洛原料药中的有关物质

Determination of related substances in cangrelor by HPLC

目的:建立坎格雷洛原料药有关物质的HPLC测定方法。方法:采用Waters Symmetry C_(18)色谱柱(250 mm×4.6 mm,5μm),以15 mmol·L^(-1)磷酸氢二铵高氯酸钠溶液(用磷酸调节pH至7.0)-乙腈为流动相,梯度洗脱;流速1.0 mL·min^(-1),检测波长242 nm;柱温30℃。结果:研究确定了坎格雷洛4个主要已知杂质,分别为A,B,C,D,其相对主成分的响应因子依次为0.590,0.510,2.27,0.660,并据此选择了杂质对照品外标法对4个已知杂质进行定量分析,对未知杂质则采用面积归一化法进行限度控制。杂质A,B,C和D均能与主成分良好分离,检测限满足控制要求,定量限浓度分别为1.00,0.048 0,0.025 0,0.075 0μg·mL^(-1),并分别在1.00~8.50,0.048 0~1.50,0.030 0~1.50,0.075 0~7.50μg·mL^(-1)范围内线性关系良好。加样平均回收率(n=9)均在90%~110%之间,RSD均小于5.0%。结论:经方法学验证,本方法既可用于坎格雷洛原料药主要工艺杂质和降解杂质A,B,C,D的准确定量,也可以对其未知杂质进行限度控制。

Objective： To establish an HPLC method for determination of the related substances in cangrelor. Methods： Waters Symmetry C15 column （250 mmx 4.6 ram,5μm） was chosen for the analysis; the mobile phase was 15 mmol·L- 1 ammonium phosphate sodium perchlorate solution （ adjusted to pH 7. 0 with phosphoric acid） -acetonitrile with gradient elution at the flow rate of 1.0 mL. min- 1 , the detection wavelength was set at 242 nm, and the column temperature was 30 ℃. Results ： The study included four main known impurities A, B, C and D in cangrelor, whose relative response factors were 0. 590, 0. 510, 2.27, 0. 660. According to this, an external stardard method using impurity reference substance was established to analyse the four impurities quantificationally, while an area normalization method was established to control the limit of unknown impurity. Chief peak and every impurity peak were separated well. Limit of Detection of A, B, C and D met the standard. Limit of Quantity of A, B, C and D were 1.00, 0. 048 0, 0. 025 0, 0. 075μg·mL^（-1） and there was a good linearity separately over the ranges 1.00-8.50, 0. 048 0 -1. 50, 0. 030 0 -1. 50, 0. 075 0 - 7. 50 μg·mL^（-1）. The average recovery （n=9） of impurity A, B, C and D was included in 90% - 110%, with RSD all less than 5. 0%. Conclusion： After methodology validation, the method can be used not only for quantitative analysis of process and degradation impurities including A, B, C and D, but for limit control of unknown impurity in cangrelor as well.