HPLC法测定2种保护组氨酸衍生物中的对映体

Determination of the Enantiomers in Two Protected Histidine Derivatives by HPLC

建立了HPLC法测定N-苄氧羰基-L-组氨酸(Cbz-L-His-OH)和1-[(1,1-二甲基乙氧基)羰基]-N-[(9H-芴基-9-甲氧基)羰基]-L-组氨酸[Fmoc-L-His(Boc)-OH]中对映体杂质的含量。色谱柱采用DAICEL CHIRALPAK■ZWIX(+)柱(3.0 mm×150 mm,3.0μm),流动相为甲醇∶水∶甲酸∶二乙胺(900∶100∶1.9∶4.16),柱温为35℃,流速为0.2 ml/min,进样量为10μl,检测波长为220 nm。结果显示,Cbz-His-OH的L和D构型、Fmoc-His(Boc)-OH的L和D构型均能达到有效分离,分离度均大于2.0;Cbz-D-His-OH和Fmoc-D-His(Boc)-OH的检测限为141、50 ng/ml,定量限为476、163 ng/ml。2对对映体在相应浓度范围内与峰面积的线性关系良好,Cbz-D-His-OH和Fmoc-D-His(Boc)-OH的加样回收率均为90.0%~100.0%,RSD(n=9)均小于3.0%。样品测定结果显示,Cbz-L-His-OH和Fmoc-L-His(Boc)-OH中对映体杂质含量均小于0.01%。建立的方法专属性强、重复性好、准确度高,可为Cbz-L-His-OH和Fmoc-L-His(Boc)-OH中对映体杂质含量的测定提供参考。

An HPLC method was established to determine the enantiomeric impurities in N-Cbz-L-histidine(Cbz-L-His-OH)and 1-[(1,1-dimethylethoxy)carbonyl]-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-histidine[Fmoc-L-His(Boc)-OH].An DAICEL CHIRALPAK■ZWIX(+)column was used,with the mobile phase of methanol∶water∶formic acid∶diethylamine(900∶100∶1.9∶4.16).The column temperature was 35℃,the flow rate was 0.2 ml/min,the injection volume was 10μl,and the detection wavelength was 220 nm.The results showed that the L-and D-enantiomers of Cbz-His-OH as well as Fmoc-His(Boc)-OH could be separated effectively.The detection and quantification limits of Cbz-D-His-OH were 141 and 476 ng/ml,for Fmoc-D-His(Boc)-OH were were 50 and 163 ng/ml,respectively.It was linear for the two pairs of enantiomers in the corresponding concentration ranges.The recoveries of both Cbz-D-His-OH and Fmoc-D-His(Boc)-OH were between 90.0% and 100.0%,and the RSDs(n=9)were less than 3.0%.The measurement results showed that contents of enantiomeric impurities in Cbz-L-His-OH and Fmoc-LHis(Boc)-OH were both less than 0.01%.The established method had strong specificity,good reproducibility and high accuracy,which provided a reference for the determination of enantiomeric impurities in Cbz-L-His-OH and Fmoc-LHis(Boc)-OH.