RP-HPLC法测定去羟肌苷肠溶胶囊有关物质次黄嘌呤及含量

RP-HPLC determination of related substance hypoxanthine and assay in didanosine enteric-coated capsules

目的:建立反相HPLC法测定去羟肌苷肠溶胶囊有关物质次黄嘌呤及含量。方法:采用Inert Surstain C_(18)(250 mm×4.6 mm,5μm)色谱柱;以0.386%醋酸铵溶液加氨水调节p H至8.0为流动相A,以乙腈-甲醇(50∶50)为流动相B,梯度洗脱,测定去羟肌苷肠溶胶囊有关物质次黄嘌呤;以流动相A-流动相B(90∶10)为流动相测定去羟肌苷肠溶胶囊中去羟肌苷含量;检测波长254 nm;流速1.0 m L·min^(-1);柱温25℃;进样体积为20μL。结果:在选定的色谱条件下,主成分及其有关物质能完全分离,杂质次黄嘌呤在0.099 6~9.960μg·m L^(-1)范围内呈良好的线性关系(r=1.000),平均回收率(n=9)为100.7%(RSD=1.9%),次黄嘌呤与去羟肌苷的相对较正因子为0.62;去羟肌苷在0.242 3~48.45μg·m L^(-1)范围内呈良好的线性关系(r=0.999 6),平均回收率(n=9)为99.9%(RSD=1.4%)。结论:本法经方法学验证,可用于去羟肌苷肠溶胶囊的有关物质检查及含量测定。

Objective： To establish a RP-HPLC method for the determination of related substance （hypoxanthine） and assay in the didanosine enteric-coated capsules. Method： An InertSurstain Cls column （ 250 mm×4.6 mm, 5 μm ） was adopted with gradient elution. Mobile phase A consisted of 0.386% ammonium acetate （ adjusted pH to 8.0 by ammonia water ） and mobile phase B consisted of methanol- acetonitrile （ 50 ： 50 ）; the assay was carried out with the mobile phase consisting of mobile phase A-mobile phase B （ 90 ： 10 ） ; the flow rate was 1.0 mL·min^-1 ; the detector wavelength was set at 254 nm; the column temperature was maintained at 25℃, and the injection volume was 20 μL. Results： The impurities and didanosine were well separated. The liner range of hypoxanthine was 0.099 6-9.960 μg·mL^-1 （ r=1.000 ）and the average recovery was 100.7% with RSD of 1.9% （ n=9 ）. The relative calibration factor of hypoxanthine with respect to didanosine was 0.62. The liner range of didanosine was 0.242 3-48.45 μg·mL^-1 （ r=0.999 6 ）, and the average recovery was 99.9% with RSD of 1.4% （ n=9 ）. Conclusion： The established method can be used for the content determination of related substances in didanosine entric-coated capsules.