HPLC法测定注射用比伐芦定的含量及有关物质

Determination of Content and Related Substances of Bivalirudin for Injection by HPLC

目的:建立测定注射用比伐芦定含量和有关物质的HPLC方法。方法:方法采用PhenomenexKinetex XB-C18(150 mm×4.6 mm, 2.6μm)色谱柱,以三氟乙酸-水-四氢呋喃-乙腈(2∶800∶100∶100)为流动相,流速0.6 ml·min^(-1),柱温50℃,检测波长210 nm,进样体积10μl。结果:在该色谱条件下,主峰与有关物质均能得到良好分离,主峰和各有关物质在测定的浓度范围内线性关系良好(r值范围0.999 1~1.000 0);比伐芦定和5种有关物质(BIVA((12-20))-BIVA、Plus-Gly-BIVA、Des-Gly-BIVA、Beta-Asp9-BIVA、Alpha-Asp9-BIVA)的检测限分别为0.238 9,0.166 4,0.297 3,0.309 1,0.312 7,0.410 7μg·ml^(-1),平均回收率分别为99.8%,99.6%,99.1%,93.1%,97.5%,102.3%,RSD分别为0.6%,4.1%,2.7%,3.4%,4.3%,1.7%(n=9~12)。结论:该方法重复性好、专属性强,可以更加有效控制注射用比伐芦定的质量。

Objective: To establish an HPLC method for the determination of content and related substances of bivalirudin for injection.Methods: The analytical column was Phenomenex Kinetex XB-C18(150 mm×4.6 mm, 2.6 μm), and the mobile phase consisted of trifluoroacetic acid-water-tetrahydrofuran-acetonitile(2∶800∶100∶100) at the flow rate of 0.6 ml·min^(-1). The column temperature was 50℃ and the detection wavelength was 210 nm. The injection volume was 10 μl. Results: Under above conditions, the main peak and the peaks of the related substances were well separated, and they all had good linear relationships(r=0.999 1-1.000 0). The detection limit of bivalirudin and five related substances(BIVA(12-20)-BIVA, Plus-Gly-BIVA, Des-Gly-BIVA, Beta-Asp~9-BIVA, Alpha-Asp~9-BIVA) was 0.238 9, 0.166 4, 0.297 3, 0.309 1, 0.312 7 and 0.410 7 μg·ml^(-1), respectively;the average recovery was 99.8%, 99.6%, 99.1%, 93.1%, 97.5% and 102.3% with the RSDs of 0.6%, 4.1%, 2.7%, 3.4%, 4.3% and 1.7%(n=9-12), respectively. Conclusion: The method is reproducible and specific, and can effectively control the quality of bivalirudin for injection.