2.3. Molecular weight (MW) measurem

2.3. Molecular weight (MW) measurement

The molecular weight distribution profiles of the samples were assessed by fast protein size-exclusion liquid chromatography (SEC-FPLC) using a Superdex™ Peptide 10/300 GL column (Amersham Biosciences; fractionation range: 7000–100 Da). The liquid chromatographic system consisted of a Waters 600 automated gradient controller pump, a Waters 717 plus autosampler and a Waters 996 photodiode array detector (Waters™, Milford, MA, USA). Data acquisition and chromatographic analyses were done with the Empower software™ program. Calibration of the column was made using standard molecules as published by Laroque et al. (2008). Nine standards of decreasing molecular weight (MW) were used for the calibration (logMW = 0.099 Rt + 5.733 where Rt is the retention time, expressed in minutes): aprotinin (6511.4 Da), insulin chain B (3495.9 Da), insulin chain A (2531.6 Da), neurotensin (1672.9 Da), substance P (1347.6 Da), luteinizing hormone releasing hormone (1182.3 Da), substance P fragment 1–7 (900 Da), leupeptin (463 Da), and glycine (75 Da). Thirty μL (30 mg mL− 1) of sterilized Maillard reaction products (MRP) were injected onto the column, and elution was carried out isocratically in Milli-Q water with trifluoroacetic acid (TFA) 0.1% and acetonitrile (70:30) at a flow rate of 0.5 mL min− 1 (Guérard, Dufossé, De La Broise, & Binet, 2001). Chromatograms were obtained at 220 nm (detection of peptide bond rearrangements), 294 nm (detection of intermediate MR compounds), and 420 nm (detection of browning) and the area under curve (AUC) obtained for each chromatogram.