超临界流体色谱法分析非对映异构体d4T-5＇-N-磷酰化苯丙氨酸甲酯

Separation of Phenylalanine Methylester 5′-Phosphoamidates of d4T Diastereoisomers by Supercritical Fluid Chromatography

采用超临界CO2流体色谱技术,分析d4T-5'-N-磷酰化苯丙氨酸甲酯手性磷的非对映异构体。色谱柱为Hpersil ODS2(250 mm×4.6 mm,5μm),流动相为夹带改性剂甲醇、乙醇和异丙醇的超临界CO2流体。以容量因子、选择性和分离度为指标,考察改性剂、背压和柱温对分离的影响。在甲醇、乙醇和异丙醇3种改性剂中,甲醇为最好的改性剂,其中在7%甲醇改性剂下,该化合物的分离度可达到3.35。在7%甲醇改性剂条件下,考察了压力(10～20 MPa)和温度(303.15～318.15 K)的影响。在优化的分离条件(改性剂为7%甲醇,流速为2 mL/min,柱温为308.15 K,背压为15 M Pa)下,d4T-5'-N-磷酰化苯丙氨酸甲酯的两种非对映异构体完全达到基线分离,分离时间约15 min。

The separation of phenylalanine methylester 5′-phosphoamidates of d4T（d4T-P-N-PheOMe） diastereoisomers has been investigated using supercritical fluid chromatography on Hypersil ODS2（250 mm×4.6 mm,5 μm）.Supercritical CO2 modified with methanol,ethanol and 2-propanol was used as mobile phase.The influence of type and concentration of alcohol modifier,back pressure and column temperature on retention factor,diastereoselectivity and resolution was studied.Among methanol,ethanol and 2-propanol,methanol was proved to be the most favorable modifier,and 7%（V/V） of methanol was the preferred concentration at which d4T-P-N-PheOMe diastereoisomers could be separated with resolution of 3.35.The effects of pressure and temperature were investigated at 7%（V/V） of methanol in the pressure range of 10-20 MPa and temperature range from 303.15 to 318.15 K.The optimal separation condition was 7% modifier in methanol as mobile phase,2.0 mL/min of flow rate for mobile phase,308.15 K of column temperature,15 MPa of back pressure.d4T-P-N-PheOMe could be separated baselinely and the running time was about 15 min under this optimal separation condition.