Multistep ultrahigh performance liquid chromatography/tandem mass spectrometry analysis for untargeted quantification of glycating activity and identification of most relevant glycation products.

The use of advanced glycation end-products (AGEs) as biomarkers for diagnosis and clinical studies is still hampered by insufficient knowledge on clinically relevant structures formed from precursors associated with defined disease states. The present study conducted untargeted analysis of the glycating activity of AGE-precursors by ultrahigh performance liquid chromatography/tandem mass spectrometry multiple reaction monitoring (UHPLC/MSMS-MRM), monitoring the loss of a nonapeptide as the glycation target. Thus, the glycating activities of seven important AGE-precursors were determined (glucose 13% and the reactive carbonyl compounds glucosone 39%, 3-deoxyglucosone 15%, 3-deoxygalactosone 26%, 3,4-dideoxyglucosone-3-ene 79%, methylglyoxal 94%, and glyoxal 97% peptide loss; 12 h/37 °C). Furthermore, UHPLC/MSMS with simultaneous precursor ion scan and information-dependent acquisition of enhanced resolution spectra and subsequent product ion scan was applied for untargeted analysis of the major AGE-structures derived from various AGE-precursors. The 20 most important modifications could be assigned to 8 AGE-structures previously reported in the literature. Seven loosely bound AGEs not yet covered by conventional methods were detected and assigned to hemiaminals. Five AGE structures did not match any known products. The method can be applied to analyze glycating activity and AGE-structures formed from various other precursors under defined reaction conditions, supporting the selection and evaluation of diagnostic AGE-markers for clinical studies.