柱前衍生化高效液相色谱法测定酶法拆分的DL半-胱氨酸含量  

Quantity Determination of Enzymatic Chiral Resolution of DL-Cysteine Using Precolumn Derivatization High Performance Liquid Chromatography

作　　者：段静静[1] 彭佳敏[1] 曹美荣[1] 李鸣[1] 侯洁[1] 

机构地区：[1]南开大学药学院,天津300071

出　　处：《南开大学学报（自然科学版）》2012年第1期46-51,共6页Acta Scientiarum Naturalium Universitatis Nankaiensis

基　　金：天津市科技支撑计划重点项目(09ZCKFSH00900)

摘　　要：建立了以1-氟-2,4-二硝基苯基-5-L-丙氨酰胺(FDAA)为手性衍生化剂、高效液相色谱法测定半胱氨酸对映异构体含量的方法.采用Inertex-C18色谱柱,以乙腈-水(含0.1%TFA)为流动相进行梯度洗脱,L-半胱氨酸、D-半胱氨酸分别在(2.50～20)mmol/L浓度范围内与峰面积呈良好的线性关系(r=0.998 3和0.998 4);其平均回收率分别为100.1%(RSD=0.67%)和99.6%(RSD=0.92%).利用上述方法对重组表达的L-半胱氨酸脱巯基酶拆分DL-半胱氨酸外消旋体混合物进行考察和优化,最终得到纯度为99.5%的D-半胱氨酸,回收率为55.54%.A high performance liquid chromatography method coupled with precolumn derivatization using 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide（FDAA） as derivative reagent has been established to determine the DL-cysteine enantiomer.Then the content of D-cysteine and L-cysteine was detected by HPLC at the condition of acetonitrile-water（containing 0.1% TFA） as mobile phase gradient elution using Inertex-C18 column.This method showed good linearity for both D-and L-cysteine ranging from 2.50～20 mmol（r=0.998 3 and 0.998 4）.The average recoveries were 100.1%（RSD=0.67%）and 99.6%（RSD=0.92%） for L-and D-cysteine respectively.In this study,DL-cysteine was resolved using expressed L-cysteine desulfhydrase,99.5% of D-cysteine with 55.54% recovery rate could be obtained by 240 min in the resolution reaction using the enzyme.

关 键 词：柱前衍生化 HPLC L半-胱氨酸脱巯基酶 D半-胱氨酸 手性拆分 

分 类 号：O657.712[理学—分析化学]