Free Nε-carboxymethyl-lysine (CML) and Nε-carboxyethyl-lysine (CEL), the most studied markers of AGE accumulation in tissues, were assessed on total liver extracts (n = 7/10 per group) after a hydrolyzing step with 0.6 M trichloroacetic acid (C2HCl3O2) and 6 M hydrochloric acid (HCl) for 12 h at 60 °C, by means of the liquid chromatography mass spectrometry (LC-MS) as previously described [33 (link)]. The chromatographic separation was performed in an UltiMate™ 3000 HPLC system (Dionex, Milan, Italy) provided by a high-resolution LTQ Orbitrap mass spectrometer (Thermo Scientific, Rodano, Italy) with an atmospheric pressure interface and an electrospray ionization (ESI) source. Liver extracts were processed through a Phenomenex Synergi reverse-phase C18 column (dimensions: 150 × 2.1 mm, particle size: 3 μm) at a flow rate of 200 μL/min. The composition of the gradient mobile phase was as follows: 95/5 to 40/60 in 25 min, 5 mM heptafluorobutanoic acid/acetonitrile. The monitored protonated molecular ions were 205.1188 m/z for CML and 379.2094 m/z for CEL. Calibration data made with CML and CEL were used for quantitative determination of the sample’s analytes.
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