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，3.0 μm)，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10 μl，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。
Abstract
An HPLC method was established to determine the enantiomeric impurities in N-Cbz-Lhistidine( Cbz-L-His-OH) and 1-[(1,1-dimethylethoxy)carbonyl]-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-Lhistidine[ 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 10 μl, 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.
关键词
高效液相色谱法 /
对映体杂质 /
N- 苄氧羰基-L- 组氨酸 /
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
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Key words
HPLC /
enantiomeric impurity /
N-Cbz-L-histidine /
1-[(1,1-dimethylethoxy)carbonyl]-N-[(9H-fluoren- 9-ylmethoxy)carbonyl]-L-histidine
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