高效液相色谱法测定芒柄花素磺酸钠有关物质

Related substance determination of formononetin by HPLC

为建立测定芒柄花素磺酸钠有关物质的方法,采用高效液相色谱法,色谱柱为Agilent HC-C_(18)(250mm×4.6mm,5μm),流动相A为乙腈,流动相B为0.01mol/L的磷酸二氢钾,流速为0.8mL/min,按照梯度洗脱,进样量为10μL,检测波长为250nm,柱温为30℃。结果表明:主药与各杂质、各杂质之间分离度良好,各杂质均能被有效检出;芒柄花素磺酸钠及杂质A,B,C,D,E,F和G的质量浓度分别在0.060~4.004(r=0.999 8),0.056~3.756(r=0.999 3),0.039~3.902(r=0.999 6),0.060~4.026(r=0.999 5),0.058~3.878(r=0.999 3),0.058~3.869(r=0.999 5),0.060~3.977(r=0.999 5)和0.040~3.952μg/mL(r=0.999 4)范围内与各自峰面积呈良好的线性关系;仪器精密度、中间精密度、稳定性试验的RSD值小于2.0%;各杂质测定的平均回收率为98.49%~101.76%,RSD值为0.37%~1.37%(n=9)。本方法专属性强、准确度高、耐用性良好,可用于芒柄花素磺酸钠有关物质的测定。

To establish a method for the related substance determination of formononetin, HPLC is performed on the column of Agilent HC-C18 （250 mm × 4.6 mm,5 μm） with the mobile phase A of acetonitrile and mobile phase B of potassium dihydrogen phosphate solution at the flow rate of 0.8 mL/min. According to gradient elute, the sample size is 10 μL, the detection wavelength is 250 nm, and the column temperature is 30 ℃. The result shows that the main drug and related substances could be well separated and detected effectively, different impurities can be detected effectively, and there is a good linear relationship between the mass concentration of formononetin and impurity A, B, C, D, E, F and G and peak area in the range of 0.060～4.004 μg/mL（r=0.999 8）, 0.056～3.756 μg/mL（r=0.999 3）, 0.039～3.902 μg/mL（r=0.999 6）, 0.060～4.026 μg/mL（r=0.999 5）, 0.058～3.878 μg/mL（r=0.999 3）, 0.058～3.869 μg/mL（r=0.999 5）, 0.060～3.977 μg/mL（r=0.999 5） and 0.040～3.952 μg/mL（r=0.999 4）, respectively. The RSD of instrument precision, intermediate precision and stability test is no more than 2.0%, and the average recovery of impurities is in the range of 98.49%～101.76% with the RSD in the range of 0.37%～1.37%（n=9）. The method is simple, reliable and accurate, and can be used to detect related substance in sodium formononetin-3＇-sulfonate.