新型脂肪胺荧光标记试剂的合成及小分子脂肪胺的衍生检测

Synthesization of novel aliphatic amine fluorescence labeling reagents and derivative determination of micromolecule aliphatic amine

目的合成一种新的以荧光素为荧光团,N-羟基琥珀酰亚胺活性酯为反应基团的脂肪胺荧光标记试剂,并建立以新试剂标记短链脂肪胺并用高效液相色谱-荧光检测法进行分离的方法。方法在p H 8.5的Na2B4O7-H3BO3缓冲溶液中,25℃下,衍生试剂浓度为50μmol/L,衍生反应进行10 min,衍生物峰面积即可达到最大且稳定。结果在优化的条件下,建立的检测方法的线性范围为0.01~2μmol/L,线性回归系数〉0.999,检测限达到0.1~1μg/L。结论衍生后的产物利用高效液相色谱-荧光检测法,可以使得6种小分子脂肪胺得到有效分离,方法具有衍生速度快,灵敏度高等优点。

Objective To synthesize a new aliphatic amine labeling reagent, using fluorescein as fluorophore and N-hydroxysuccinimidyl acetive ester as reaction group, and establish a separation method by labeling short chain aliphatic amine with new reagent and using high performance liquid chromatography- fluorescence detection. Methods The derivatization reaction was achieved with the buffer of Na2B407-H3BO3 at pH 8.5, the reaction temperature at 25 ＊C, the reagent solution at 50 ~mol/L and the reaction time of 10 min. Results The linear range from 0.01 ~mol/L to 2 pmol/L was observed for the proposed method. The limit of detection （LOD） of developed method was between 0.1 gg/L and 1 gg/L with the linear regression coefficient above 0.999. Conclusion The baseline separation of 6 small molecule aliphatic amine derivatives is achieved using optimized HPLC fluorescence detection. The method has the advantages of high derivative speed and sensitivity.