柱前衍生化RP-HPLC荧光色谱法分离肌肽的光学异构体

Enantiomeric separation of carnosine by fluorescence RP-HPLC with pre-column derivatization

目的采用柱前衍生化反相高效液相荧光色谱法拆分肌肽的对映体。方法采用邻苯二甲醛和N-乙酰-L-半胱氨酸为柱前手性衍生化试剂,反相液相色谱法分离肌肽的光学异构体。考察衍生化反应条件对肌肽衍生化反应、不同色谱条件下对肌肽光学异构体分离的影响。通过光学异构体分离过程中的热力学参数的计算,探讨光学异构体分离过程的驱动力。结果邻苯二甲醛和N-乙酰-L-半胱氨酸的摩尔比为1∶2时,以无水乙醇与硼酸缓冲液(pH9.8)为溶剂溶解,作为衍生化试剂,于室温下避光反应5 min,肌肽转化成异吲哚产物;在柱温35℃时,肌肽光学异构体的衍生化产物分离因子(α)和分离度(R)分别为1.533、3.947。其光学异构体-衍生产物的拆分过程主要是一个焓驱动的过程。结论所用方法简单快速、灵敏度高、重复性好,适用于肌肽的手性拆分。

OBJECTIVE To establish fluorescence RP-HPLC for enantioseparation of carnosine by pre-column derivation.METHODS The separation of carnosine was studied using o-phthalaldehyde（OPA） and N-Acetyl-L-aminothiopropionic acid（NAC） as derivatizing reagents.The effects of the derivatizing procedure on the reaction,and the various chromatographic conditions on the enantioseparation were investigated.Through calculation of thermodynamic parameters in the process of enantiomeric separation,the driving force was explored.RESULTS When the derivating agent,OPA and NAC molar ratio was 1：2,dissolved by dehydrated alcohol and boric acid（PH9.8）,the carnosine was transformed to isobenzopyrrole in 5 min with the reaction away from light of the room temperature.When the column temperature was 35 ℃,the separation factor and resolving power of the carnosine diastereomers derivation production were 1.533 and 3.947.The process of the diastereomers derivation was enthalpy driving.CONCLUSION The method is simple,fast,accurate for the chiral separation of carnosine enantiomers.