荧光标记-高效液相色谱测定多肽（PC2～PC6）的研究

摘要

基于多肽（Polypeptides PC2～ PC6）中富有巯基（-SH）官能团，其与单溴二胺（Monobromobimanes mBBr）能够发生缩合反应，生成具有荧光信号的多肽衍生物；通过优化色谱分离条件，建立了高效液相色谱（荧光检测器）测定多肽的方法；试验中利用质谱鉴定了PCs与mBBr缩合反应的比例关系。结果显示，通过比较PCs标记前后化合物的质谱图，PCs与 mBBr反应比例关系为1∶1，稳定性好；高效液相色谱-荧光法测定多肽化合物，五种化合物间分离度较好，出峰时间集中在16.6～22.0 min间；PC2，PC3，PC4，PC5和PC6线性相关性系数＞0.9991，方法定量限分别为0.3，0.05，0.3，0.5和0.8 mg・ L －1，回收率范围为83.0％～102.0％；重线性较好，RSD＜2.0％，该方法实现了多肽类化合物快速、准确定量分析。%This study was based on the thiol groups(-SH) of PC2 ~ PC6 ,which could be reacted with the Monobromobimane (mBBr) ,in order to get polypeptide derivatives with fluorescent signal .A new method was developed for measuring the Poly-peptides by high performance liquid chromatography with fluorescence detector ,then the chromatographic conditions of HPLC was optimized ;meawhile the reaction proportion of PCs and mBBr was identified by Trap-MS .The results showed that ,the re-action proportion of PCs and mBBr was 1∶1 ,the polypeptide derivatives had good stability ;the five compounds separation was better ,and the peak time focused on the 16.6~22.0 min;the linear correlation coefficient of PC2 ,PC3 ,PC4 ,PC5 and PC6 was>0.999 1 ,and the limits of quantification were 0.3 ,0.05 ,0.3 ,0.5 and 0.8 mg ・ L -1 respectively ,the recovery rate was 83.0% ~102.0% ;the method was reproducible ,RSD<2% ,this method for measuring the peptide compounds was rapid and accurate .