采用同位素稀释液相色谱-电喷雾串联质谱法分析乳制品中的晚期糖基化终产物。以羧甲基赖氨酸为例。
Analysis of advanced glycation endproducts in dairy products by isotope dilution liquid chromatography-electrospray tandem mass spectrometry. The particular case of carboxymethyllysine.
作者信息
Delatour Thierry, Hegele Jörg, Parisod Véronique, Richoz Janique, Maurer Sarah, Steven Matthew, Buetler Timo
机构信息
Nestlé Research Centre, Nestec Ltd, Vers-chez-les-Blanc, Lausanne, Switzerland.
出版信息
J Chromatogr A. 2009 Mar 20;1216(12):2371-81. doi: 10.1016/j.chroma.2009.01.011. Epub 2009 Jan 15.
A fully validated multiple-transition recording isotope dilution liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantitative determination of N(epsilon)-carboxymethyllysine (CML) and lysine in dairy products is described. Internal standards were [N-1',2'-(13)C(2)]CML and [1,2,3,4,5,6-(13)C(6)-2,6-(15)N(2)]lysine, and the method was validated by evaluating the selectivity, linearity, precision (repeatability and reproducibility) and trueness, using both powder and liquid products. For liquid dairy products, the repeatability and reproducibility was 2.79% and 11.0%, while 4.85% and 4.92% were determined for powder dairy products, respectively. The trueness of the method ranged from -9.6% to -3.6% for powder and from -0.99% to 6.8% for liquid dairy products. The limit of detection for CML was estimated to be 8 ng CML per mg protein while the limit of quantification was 27 ng CML per mg protein. The method encompasses a proteolytic cleavage mediated by enzymatic digestion to reach a complete release of the amino acids prior to a sample cleanup based on solid phase extraction, and followed by LC-MS/MS analysis of CML and lysine residues. To ensure a suitable performance of the enzymatic digestion, CML measurements were compared to values obtained with an acid hydrolysis-mediated proteolysis. Finally, the method was employed for the analysis of CML in various dairy products. The values compare well to the data available in the literature when similar methods were used, even if some discrepancies were observed upon comparison with the results obtained by other techniques such as enzyme-linked immunosorbent assay and GC-MS.
描述了一种经过充分验证的多转换记录同位素稀释液相色谱 - 电喷雾串联质谱法(LC-MS/MS),用于同时定量测定乳制品中的N(ε)-羧甲基赖氨酸(CML)和赖氨酸。内标物为[N-1',2'-(13)C(2)]CML和[1,2,3,4,5,6-(13)C(6)-2,6-(15)N(2)]赖氨酸,该方法通过评估选择性、线性、精密度(重复性和再现性)和准确性进行验证,使用了粉末状和液态产品。对于液态乳制品,重复性和再现性分别为2.79%和11.0%,而粉末状乳制品的重复性和再现性分别为4.85%和4.92%。该方法的准确性对于粉末状乳制品范围为-9.6%至-3.6%,对于液态乳制品范围为-0.99%至6.8%。CML的检测限估计为每毫克蛋白质8纳克CML,定量限为每毫克蛋白质27纳克CML。该方法包括酶促消化介导的蛋白水解,以在基于固相萃取的样品净化之前实现氨基酸的完全释放,然后对CML和赖氨酸残基进行LC-MS/MS分析。为确保酶促消化具有合适的性能,将CML的测量值与酸水解介导的蛋白水解获得的值进行了比较。最后,该方法用于分析各种乳制品中的CML。当使用类似方法时,这些值与文献中的数据比较良好,即使在与酶联免疫吸附测定和气相色谱 - 质谱等其他技术获得的结果进行比较时观察到一些差异。