HPLC测定FLT3激酶抑制剂LY266及其稳定性

Determination and stability of the FLT3 kinase inhibitor LY266 by HPLC

目的测定FLT3激酶抑制LY266的酶及细胞水平的活性和含量，并考察其稳定性。方法体外FLT3激酶抑制分析采用Merck Millipore的Kinase Profiler完成；细胞增殖抑制试验采用MV4—11细胞系测定；稳定性采用Venusil XBP—C18色谱柱（250mm×4．6mm，5μm），流动相为甲醇-水（75：25），检测波长247．6nm，流速1．0mL·min^-1，柱温30℃。结果酶水平的活性和细胞增殖抑制的IC50分别为20．56、0．1156nmol·L^-1；LY266能与相邻杂质完全分离，4～120μg·mL^-1LY266与峰面积的线性关系良好（r=0．9991）；高、中、低浓度精密度试验的RSD分别为1．05％、0．36％、1．14％；平均回收率分别为100．80％、99．91％、100．51％，RSD分别为0．83％、1．21％、1．14％；重复性试验的RSD=0．39％。LY266在高温、高湿和强光下均较稳定。结论所用方法的专属性强、灵敏度高，可用于测定LY266的含量和稳定性研究。

OBJECTIVE To determine the enzyme level and cell level activity of FLT3 kinase inhibitor LY266,and to investigate its stability. METHODS In vitro FLT3 kinase inhibition was performed using the Kinase Profiler service provided by Merck Millipore. Cell proliferation inhibition assay was performed using the MV4 - 1 l cell line. The Venusil XBP - C18 （250 mm × 4.6 mm, 5 μm） column was used. The mobile phase was methanol - water （75 ： 25 ） with the flow rate of 1.0 mL· min ^-1. Wavelength was 247.6 nm of the UV and the column temperature was 30 ℃. RESUITS The IC50 value of the enzyme level activity was 20.56 nmol· L^-1. The IC50 value for cell proliferation inhibition was 0. 1156 nmol· L^-1. LY266 can be completely separated from the adjacent impurities. LY266 had a good linear relationship with the peak area （ r = 0. 9991 ） in the range of 4 - 120 μg· mL^- 1. The RSD values of the high, medium and low concentration precision tests were 1.05% ,0.36% , and 1.14%. The average recovery and RSD were 100.80% ,99.91% , 100. 51% and 0.83%, 1.21%, 1. 14%, respectively. The reproducibility was 0.39%. The result of influencing factors showed that LY266 in high temperature, high humidity and light were more stable. CONCLUSION The method used is strong and sensitive, and it is suitable for the determination and stability of LY266.