Highly-Sensitive Detection of Free Advanced Glycation End-Products by Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry with 2,4,6-Trinitrobenzene Sulfonate Derivatization
2013
Hashimoto, Chihiro | Iwaihara, Yuri | Chen, Si Jing | Tanaka, Mitsuru | Watanabe, Toshiaki | Matsui, Toshiro
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 of >1000 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.
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