基于蛋白沉淀法超高效液相色谱-串联质谱法检测面包中两种蛋白结合型晚期糖基化终末产物

Determination of two protein-bound advanced glycation end-products in bread by ultra-high performance liquid chromatography-tandem mass spectrometry based on protein precipitation

目的建立面包中两种典型晚期糖基化终末产物(AGEs)的超高效液相色谱-串联质谱检测方法。方法以结合型羧甲基赖氨酸(CML)和羧乙基赖氨酸(CEL)为检测对象,面包样品经还原孵育、蛋白沉淀、加同位素内标、酸水解、氮吹、九氟戊酸水溶液复溶后,多反应监测定性及定量分析。结果方法的检出限CML为4.5 ng/g,CEL为0.5 ng/g;定量限CML为20 ng/g,CEL为2 ng/g;CML加标回收率为89.62%~95.65%,CEL的加标回收率为86.38%~97.17%;标准曲线线性范围为CML 2.5~800 ng/mL和CEL 0.25~80 ng/mL,决定系数均大于0.999。结论该方法准确且灵敏,能够满足面包中AGEs的检测需求。

Objective To establish an ultra-high performance liquid chromatography-tandem mass spectrometry method for the detection of two typical advanced glycation end products(AGEs)in bread.Methods Protein-bound carboxymethyl lysine(CML)and carboxyethyl lysine(CEL)were used as the detection objects.Bread sample was subjected to reduction incubation,protein precipitation,addition of isotope internal standard,acid hydrolysis,nitrogen blowing,reconstitution with nonafluorovaleric acid aqueous solution,and then underwent multi-reaction monitoring to quantitative analysis.Results The detection limit and quantification limit of the method were 4.5 ng/g and 20 ng/g for CML,and 0.5 ng/g and 2 ng/g for CEL.The spike recovery rates of CML and CEL were 89.62%-95.65%and 86.38%-97.17%.The linear range were 2.5-800 ng/mL and 0.25-80 ng/mL,and the coefficients of determination were both above 0.999.Conclusion The method was accurate and sensitive,and could meet the detection requirements of AGEs in bread.