对硝基苯乙二胺衍生化-HPLC检测脂肽方法

The nitrobenzene ethylenediamine derivatives-HPLC method to measure the fat peptide

脂肽中含有酰胺键、酯键等双键结构,其最大紫外吸收波长位于205 nm左右,溶剂效应及绝大部分物质在此波长附近都有吸收,因此,直接使用HPLC法检测脂肽的相对标准偏差大,溶剂效应及杂质干扰导致检测灵敏度及准确率下降。针对该问题,提出了对硝基苯乙二胺衍生化-HPLC检测脂肽的新方法,通过对硝基苯乙二胺衍生后的Surfactin,再用HPLC检测,则有色谱信号最强、峰面积更大、峰宽更窄且拖尾现象较弱、衍生率高、不易漏检低含量的脂肽等优点,能够降低脂肽的检出限。利用标准曲线的斜率和色谱噪音及仪器信噪比确定了对硝基苯乙二胺衍生化Surfactin的检出限为6.75×10^(-2)mg/L、相对标准偏差在3%以内、样品回收率为99%~101%,表明该衍生化方法具有良好的重复性,检测方法准确率高,适于Surfactin的微量检测。

For fat peptide containing amide linkage,ester bond,such as double bond structure,its biggest ultraviolet absorption is at 205 nm,solvent effect and the vast majority of material near the wavelengths are absorbed,HPLC method directly due to the interference of impurity in the sample relative standard deviation is large,resulting in a decline in the detection sensitivity and accuracy.To solve these problems,nitrobenzene ethylenediamine derivatives-a new method of HPLC to detect lipid peptide was put forward.The method changed the maximum absorption wavelength of Surfactin and improved the response value of ultraviolet,based on the nitrobenzene ethylenediamine tag Surfactin chromatographic signals,the strongest derivative Surfactin peak area was bigger,peak width was narrower and trailing phenomenon was weaker,derivative rate was high,not easy to leak peptide,low content of fat could reduce the detection limit of lipid peptide.With the slope of standard curve and the chromatographic and instrument noise SNR,the p-nitrophenyl ethylenediamine derivatives Surfactin detection limit of 6.75×10^(-2)mg/L was determined,relative standard deviation was within 3%,sample recovery rate was in 99%-101%,which showed that the derivative method had good repeatability,high detection accuracy,suitable for Surfactin trace detection.