Zb 1701

MCF derivatization of balsamic vinegars was carried out to determine the profile of amino and non-amino organic acids, and some primary amines and alcohols. The sample preparation protocol was adopted from that of Pinu et al. [30 (link)] and then optimized for the analysis of vinegars. An amount of 130 µL of vinegar was mixed with 20 µL internal standard l-alanine-2,3,3,3-d₄ (10 mM) and 50 µL of NaOH (2 M) in silanized reaction tubes. MCF derivatization was performed according to the method of Smart et al. [27 (link)] After derivatization, all the samples were injected into an Agilent GC 7890 coupled to an Agilent MSD 5975 with a quadrupole mass selective detector (Electron Ionization; positive mode) operated at 70 eV. The GC column used for all analyses was a Zebron ZB-1701 (Phenomenex, Torrance, CA, USA), 30 m × 250 µm (internal diameter) × 0.15 µm (film thickness), with a 5-m guard column. The MS was operated in scan mode (start after 6 min; mass range 38–650 a.m.u. at 1.47 scans/s). All the other analytical parameters are described in Smart et al. [27 (link)].

The manual TMS derivatisation was performed following the protocol published in Pinu et al. [21 (link)] and the derivatised samples were injected into an Agilent GC 7890 coupled to a MSD 5975 (Agilent Technologies, Santa Clara, CA, USA) with a quadrupole mass selective detector (Electron Ionisation) operated at 70 eV. The column used for the analysis of TMS-derivatized samples was a Zebron ZB-1701 (Phenomenex, Torrance, CA, USA), 30 m × 250 µm (internal diameter) × 0.15 µm (film thickness), with a 5-m guard column. The MS was operated in scan mode, where scanning started after 5 min (mass range 40 to 650 atomic mass unit (a.m.u ) at 1.47 scans/s). GC-MS parameters are already described in Villas-Boas et al [18 (link)].

Endogenous glucose production was determined as described previously with minor modifications (van Dijk et al. 2013): Food was removed at 9 AM and the experiment started at 1 PM. At 1 PM, animals received 0.1 mg/g [6,6]‐D2‐glucose intraperitoneally at a concentration of 30 mg/mL. Immediately before, and 10, 20, 30, 40, 50, 60, 75, and 90 min after the intraperitoneal injection, a small amount of blood was collected on a filter paper and glucose measured with an Accu‐Check^® glucose meter. Glucose was then extracted from the dried blood with a water/ethanol mixture. After the solution was evaporated under nitrogen flow at 60°C, the residue was derivatized to glucose penta‐acetate by adding 100 μL pyridine and 200 μL acetic anhydride to the extracted glucose and heating at 60°C for 30 min. After evaporation under nitrogen flow, the residue was then dissolved in 200 μL ethyl acetate for analysis by GC–MS. GC–MS was performed with a Zebron ZB‐1701 30 m × 250 μm × 0.25 μm (Phenomenex) column under positive chemical ionization with ammonia ions monitored at m/z 408–412 (m₀–m₄). After corrections for natural abundances according to Lee et al., the kinetics of the enrichment of glucose with D2‐glucose were then used to estimate endogenous glucose production and related parameters according to van Dijk et al. (2013).