Search

The purpose of this study was to gain insight into the production behavior of free adducts of advanced glycation end-products (AGEs) in Wistar rats under acute hyperglycemic conditions. Five AGE-free adducts as well as their precursors (i.e., highly reactive carbonyl intermediates of methylglyoxal and glyoxal) in rat plasma were quantitatively determined at greater than nanomolar levels using the liquid chromatography/tandem mass spectrometry method coupled with 2,4,6-trinitrobenzene sulfonate and 2,3-diaminonaphthalene derivatization techniques. An oral glucose (2 g/kg dose) tolerance test to 10-week-old Wistar rats provided evidence that the plasma levels of diabetes-related metabolites did not change acutely within 120 min, irrespective of increasing blood glucose levels.

The common derivatization method of primary amino groups by 2,4,6-trinitrobenzene sulfonate (TNBS) was applied to the detection of free adducts of advanced glycation end-products (AGEs), in combination with liquid chromatography-tandem mass spectrometry-multiple reaction monitoring (LC-MS/MS-MRM). The proposed TNBS-MS method provided a surprisingly significant improvement of the detection of AGE-free adducts (e.g., by a factor of1000 for methylglyoxal-derived hydroimidazolone) with a detection limit of 1.0 nM (10 fmol/injection volume), which was due to the high ionization efficiency of the derived trinitrophenol moiety and its hydrophobicity. With the aid of stable-isotope-labeled internal standard (MG-H1-d3), the convenient TNBS method that allowed the derivation of AGE-free adducts bearing amino groups under mild reaction conditions (30 mM, pH 8.5, 30 min, 30 °C) also permitted successive detection and quantification of five typical AGE-free adducts at nanomolar levels in rat plasma (50 μL) with high reproducibility (2-9% of RSD) and recovery (93-113%), using LC-MS/MS-MRM.