Synthesis, Purification, and Mass Spectrometric Characterization of Stable Isotope-Labeled Amadori-Glycated Phospholipids

[Image: see text] Nonenzymatic glycation of lipids plays an important role in several physiological and pathological processes, such as normal aging and complications of diabetes mellitus. To develop liquid chromatography coupled with mass spectrometric (LC-MS) methods for accurate analysis of Amadori compound-glycated lipids from biological samples, it is essential to obtain isotope-labeled Amadori-lipid standards. Herein, we report optimized methods for the preparation of six stable isotope-labeled Amadori-glycated lipid standards covering four types of lipids, including [(13)C(6)]Amadori-phosphatidyl ethanolamine (PE), -phosphatidyl serine (PS), -LysoPE, and -LysoPS. Optimal conditions for the synthesis and purification of these four types of Amadori-glycated lipids were detailed in this study. LC-MS and LC-UV analyses showed that destination products were highly purified (>95%). Accurate mass and MS/MS fragmentation in both positive- and negative-ion modes further validated the identification of these six synthetic [(13)C(6)]Amadori-glycated lipid standards. Successful preparation of these highly purified isotope-labeled standards makes it possible to develop targeted LC-MS/MS methods for accurate analysis of Amadori-glycated phospholipids from biological samples.