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

Abstract

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 [13C6]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 [13C6]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.

Comprehensive Identification of Amadori Compound-Modified Phosphatidylethanolamines in Human Plasma

“…First, LC-NLS-MS was performed to scan precursor ions, which had neutral loss of 303 Da in positive mode and 162 Da in negative mode. These two neutral loss are characteristic of Amadori-PE/LPE as reported previously. , LC-NLS-MS in positive and negative modes led to two lists of possible Amadori-PE/LysoPE species. Species shared by the two lists were defined as candidate Amadori-PE/LysoPE species.…”

“…Okudaira et al used acidic MeOH (pH 4.0) to extract lysophospholipids from mouse plasma, serum, and tissues, and their work showed that the relatively lower pH could help to inhibit the migration of acyl chain in lysophospholipids from sn -2 to sn -1 position . Alternatively, we used phenyl boronate affinity (PBA) resin to separate Amadori-PE from PE during synthesis of Amadori-PE standards …”

“…For verification and more specific fatty acyl sn -position identification, LC-PIS-MS was further performed to obtain molecular fragment information on these candidate species. Fragmentation patterns of Amadori-PE/LysoPE had been obtained in our previous study, which made it possible to exclude false identifications that were not conforming to the common patterns (Figure ).…”

“…Amadori-PE/LysoPE standards (Amadori-PE­(15:0/15:0), -PE­(16:0/18:1­(9Z)), and -LysoPE­(13:0/0:0)) were synthesized by incubation of their respective substrates with d -glucose in anhydrous MeOH, as described previously . Briefly, for synthesis of Amadori-PE, 7 μmol of PE substrates were mixed with 161 μmol of d -glucose in 1 mL of MeOH and, then, stirred at 600 rpm in a Thermomix R Mixer (Eppendorf, Germany) at 60 °C for 3 days; for synthesis of Amadori-LysoPE, 12 μmol of LysoPE substrate was mixed with 215 μmol of d -glucose in 1 mL of MeOH, and stirred at 50 °C for 3 days.…”

“…Amadori-PE(15:0/15:0), -PE(16:0/18:1(9Z)), and -LysoPE(13:0/ 0:0) from synthesis were purified through boronic affinity SPE and preparative HPLC separation in order, as described previously. 19 Briefly, Amadori-PE/LysoPE were separated from their phospholipid substrates by boronate affinity SPE (Bond Elut PBA cartridges, 500 mg, Agilent); then, they were further purified with a Kromasil C18 column (250 × 10 mm, 10 μm, AkzoNobel, Bohus, Sweden) on a Shimadzu 20A HPLC. Isocratic mobile phases of 100% and 80% MeOH both containing 5 mM ammonium formate were used for Amadori-PE and Amadori-LysoPE, respectively, at a flow rate of 3 mL/min.…”