2.7. Determination of Amino Acids Using LC-MS

One gram of bread sample, in triplicates, with a mass of 45 ± 0.1 mg, was added to 1.40 mL volume of 50% methanol containing 10 mg L⁻¹ of 2,3,3,3-d4-alanine (d4A) in a microcentrifuge tube. The tubes were vortexed to obtain a homogenous mixture. This was followed by centrifugation at 10.000 rpm for 5 min at 4 °C (Z216MK, HERMLE Labortechnik GmbH, Wehingen, Germany). A 10 µL volume of extract was used to perform precolumn derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, following a method adapted from Salazar et al. [50].

The LC-MS was an Agilent 1260 Series liquid chromatograph comprising a G1311B quaternary pump, G1329B thermostatted autosampler and a G1330B thermostatted column compartment (Agilent Technologies, Santa Clara, CA, USA). The mobile phase A was 0.6% formic acid in ultrapure water and mobile phase B was 0.1% formic acid in acetonitrile. A Phenomenex Kinetex EVO C18 column was used, measuring 2.1 × 150 mm with 1.7 µm diameter packing material, maintained at 25 °C. The chromatographic gradient was held for 1 min at 1.5% B and then ramped to 13% B at eight min, 17% B at 15 min and 80% B at 16 min, before returning to 1.5% B at 17.5 min. The flow rate was 300 µL/min, total run time was 28 min, and the injection volume was 5 µL. For detection, an Agilent 6420 triple quadrupole mass spectrometer fitted with an Agilent Multimode Ionization source was operated in positive electrospray mode. Optimum Multiple Reaction Monitoring (MRM) transitions were established using Agilent Mass Hunter Optimizer B06.00 software (Agilent Technologies, Santa Clara, CA, USA). Derivatized amino acid peak areas were normalized to the recovery of d4A and quantified by reference to a dilution series of external standards prepared from a commercial amino acid mix (Sigma product A9906, Sigma-Aldrich Pty. Ltd., Sydney, Australia). Data were collected and processed using the Agilent Mass Hunter software.