目的: 建立高效液相色谱同时测定N-芴甲氧羰基-O-叔丁基-L-苏氨酸中6个特定杂质的方法。方法: 采用YMC Triart C18(250 mm×4.6 mm,3 μm)色谱柱,以0.1%三氟乙酸水溶液为流动相A,以0.1%三氟乙酸乙腈溶液为流动相B,流速1.0 mL·min-1,梯度洗脱,检测波长265 nm,柱温30 ℃,进样体积10 μL。结果: N-芴甲氧羰基-O-叔丁基-L-苏氨酸与相邻杂质峰的分离良好;6个杂质分离度均大于1.5;且在相应质量浓度范围内呈现良好的线性关系(r≥0.999);6个杂质检测限和定量限分别约为0.03 μg·mL-1和0.06 μg·mL-1;6个杂质的平均回收率(n=9)在97.6%~98.8%范围内。3批N-芴甲氧羰基-O-叔丁基-L-苏氨酸测定结果显示,杂质1的含量<0.2%,杂质4的含量<0.1%,其他4种杂质未检出,总杂含量<1%。结论: 本方法分离度好,灵敏度高,专属性强,适用于N-芴甲氧羰基-O-叔丁基-L-苏氨酸中有关物质的检测。
Objective: To establish an HPLC method for the determination of six related substances in N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonine. Methods: The analysis was conducted on YMC Triart C18(250 mm×4.6 mm, 3μm) column, the mobile phase was consisted with 0.1% trifluoroacetic acid in water(A) and 0.1% trifluoroacetic acid in acetonitrile(B)at the flow rate of 1.0 mL·min-1. The column temperature was set 30 ℃, the detection wavelength was 265 nm and the injection volume was 10 μL. Results: N-Fluorenylmethoxycarbonyl-O-tert-butyl-L-threonine had good separation from the adjacent impurity peaks; The resolution of impurity1- 6 was greater than 1.5, and showed a good linear relationship (r≥0.999) in the corresponding mass concentration range, the detection limit of impurity 1-6 was 0.03 μg·mL-1, and the quantitative limit was 0.06 μg·mL-1, the average recovery rate (n=9) of impurity 1-6 was in the range of 97.6%-98.8%. The results of the three batches of N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonine showed that the contents of impurity 2, impurity 4 were <0.2% and <0.1%, respectively, and the other four impurities were not detected, and content of the total impurity was <1%. Conclusion: This method has good resolution, high sensitivity and strong specificity, and is suitable for the determination of related substances in N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonine.
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