HPLC法测定盐酸替罗非班原料药的含量及有关物质

Content Determination of Tirofiban Hydrochloride and Its Related Substances by HPLC

目的：建立测定盐酸替罗非班原料药含量及有关物质的方法。方法：采用高效液相色谱法。色谱柱为CAPCELL PAK C18,流动相A为0.1%醋酸铵溶液（p H 2.30）-乙腈-甲醇（90∶5∶5,V/V/V）、流动相B为0.1%醋酸铵溶液（p H 2.30）-乙腈-甲醇（10∶85∶5,V/V/V）（梯度洗脱）,检测波长为227 nm,柱温为35℃,流速为1.0 ml/min,进样量为10μl。结果：盐酸替罗非班与其中间体及其他杂质分离度良好。盐酸替罗非班的质量浓度在20~140μg/ml范围内与其峰面积积分值呈良好的线性关系（r=0.999 9）;精密度、稳定性、重复性试验的RSD≤0.35%;平均回收率为100.09%,RSD=0.81%（n=3）;其中间体的检测限、定量限分别为0.5、2.0ng。盐酸替罗非班原料药中的单个杂质含量低于0.05%,总杂质含量低于0.2%。结论：该方法简便、准确、专属性强、灵敏度高,适用于盐酸替罗非班原料药的含量及有关物质测定。

OBJECTIVE ： To establish a method for the content determination of tirofiban hydrochloride and its related substances. METHODS： HPLC method was adopted. The determination was performed on CAPCELL PAK C18 column with mobile phase A consisted of 0.1% ammonium acetate （pH2.30）-acetonitrile-methanol （90 ： 5 ： 5, V/V/F） and mobile phase B consisted of 0.1% ammonium acetate （pH 2.30）-acetonitrile-methanol （10：85：5, V/V/V）gradient elution at the flow rate of 1.0 ml/min. The detection wavelength was set at 227 nm, and the column temperature was 35 ℃. The sample size was 10 μl. RESULTS： The main peak and intermediates as well as individual impurity peaks were well separated. The linear range of tirofiban hydrochloride was 20-140 gg/ ml （r=0.999 9） with an average recovery of 100.09% （RSD=0.81% ,n=3）. RSD of precision, stability and reproducibility tests were all ≤0.35%. The detection limit and quantification limit of intermediates were 0.5 ng and 2.0 ng. The content of individual impurity in tirofiban hydrochloride was lower than 0.05%, and that of total impurities was lower than 0.2%. CONCLUSIONS： The method is simple, accurate, specific and sensitive, and it is suitable for the content determination of tirofiban hydrochloride and its related substances.