*2.8. LC-ESI-MS/MS Optimized Analytical Conditions*

Acrylamide determination was carried out through liquid chromatography coupled with mass spectrometry using an Agilent Technologies RP-LC-ESI-MS/MS triple quad system (Santa Clara, CA, USA), consisting of an HPLC (Agilent Technologies 1200 Series) equipped with a degasser, a binary pump, an autosampler, and a 6410B triple quadrupole mass spectrometer.

The MS/MS parameters (parent ion, product ion, fragmentor, collision energy, and polarity of the ESI ion source) were optimized using standard solutions (0.125 mg/L) of acrylamide and acrylamide d3 through their individual introduction into the MS source. Selected reaction monitoring (SRM) transitions of the most abundant fragments (product ions) were used.

Vials containing the samples were then loaded into an autosampler carousel at a controlled temperature (20 ◦C), and 5 μL of each sample were injected into a Gemini RP C18 column (Phenomenex, Torrance, CA, USA) (25 cm × 2 mm i.d. × 5 μm particle size × 110 Å pore size). The solvent system was composed of water with formic acid 0.1% (solvent A) andacetonitrile with formic acid 0.1% (solvent B). The elution (17 min of run + 10 min of post-run) was carried out according to the following gradient: 0% B (6.5 min), 95% B (7.5 min), 95% B (13.0 min), 0% B (13.1 min), 0% B (17.0 min) with a flow rate of 0.23 mL/min at a controlled temperature of 20 ◦C (pressure of about 180 bars at run start).

Source parameters were optimized as follows: gas (N2) temperature, 300 ◦C; N2 flow, 3 L/min; nebulizer pressure, 30 psi; capillary, positive 2500 V and negative 1500 V. Peak identification included comparison of peak retention times to those obtained with calibrants and evaluation of the tandem mass spectroscopic experiments. Quantification was performed by external standard calibration in the presence of acrylamide d3 as the internal standard, and the chromatograms were processed using MassHunter Workstation Quantitative Analysis software vB05.00 (Agilent Technologies Inc., Santa Clara, CA, USA).
