Determination of six related substances in N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonine by HPLC
Objective:To establish an HPLC method for the determination of six related substances in N-fluore-nylmethoxycarbonyl-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-Fluorenylme-thoxycarbonyl-O-tert-butyl-L-threonine had good separation from the adjacent impurity peaks;The resolu-tion of impurity 1-6 was greater than 1.5,and showed a good linear relationship(r≥0.999)in the correspond-ing 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-fluorenyl-methoxycarbonyl-O-tert-butyl-L-threonine.
N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonineHPLCthe six impuritiesgradient elution
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