HPLC测定硫唑嘌呤中有关物质

Determination of Related Substance in Azathioprine by HPLC

目的建立用HPLC测定硫唑嘌呤中有关物质的测定方法。方法采用Phenomenex C18(4.6mm×200mm,5μm)色谱柱,流动相为甲醇-0.05%醋酸钠水溶液(18∶82),流速:1.2mL·min-1,检测波长240nm。结果在选定的色谱条件下,硫唑嘌呤与起始物料、中间体等有关物质完全分离;巯嘌呤在0.45～1.125μg·mL-1(r=0.9999),1-甲基-4-硝基-5-氯咪唑(硝化物)在0.3～0.75μg·mL-1(r=0.9999)内呈良好的线性关系;巯嘌呤和1-甲基-4-硝基-5-氯咪唑平均回收率(n=9)分别为100.1%和99.7%;检测限分别为3ng.mL-1和2ng·mL-1。结论本测定方法简便、准确、专属性好,可用于硫唑嘌呤的有关物质的测定。

OBJECTIVE To establish an HPLC method for the determination of related substance in Azathioprine. METHODS Phenomenex C18 （4.6 mm×200 mm,5 μm ） column was used, the mobile phase consisted of methanol-0. 05% sodium acetate（ 18 ：82） , at flow rate of 1.2 mL·min^-1, detection wavelength was set at 240 nm. RESULTS On the selection chromatogramic condition, related substances of Azathioprine, starting material, intermediate and etc were separated completely. The linear ranges were 0.45 - 1. 125 μg·mL ^-1 （r =0. 999 9） for Mercaptopurine and 0.3 -0.75 μg·mL^-1 （r =0. 999 9） for 5-chloro-l-methyl-4- Nitroimidazole. The average recoveries（ n = 9） of Mercaptopurine and 5-chloro-l-methyl-4-Nitroimidazole were 100.1% and 99.7% respectively. The limit of quantitations were 3 ng·mL^-1 and 2 ng·mL^-1. CONCLUSION The method proposed for the determination of the related substances of Azathioprine is simple, accurate and specific.