2.2. Derivatization Procedures A and B for Amino Acids and Generation of GC-MS Spectra

Procedure A. Solid amino acids were derivatized first with 2 M HCl/CH₃OH or 2 M HCl/CD₃OD and then with PFPA/EA in autosampler glass vials. Briefly, residues were reconstituted in 100 µL aliquots of a 2 M HCl/CH₃OH or 2 M HCl/CD₃OD solution and the glass vials were tightly sealed. Esterification was performed by heating the samples for 60 min at 80 °C. After cooling the samples of the esterification reaction to room temperature, solvents and reagents were evaporated to dryness under a stream of nitrogen. Aliquots (100 µL) of the PFPA/EA solution were added, and the glass vials were tightly sealed and heated for 30 min at 65 °C to prepare N-pentafluoropropionic amides of the methyl esters. Then, residues were treated first with 200 µL aliquots of 400 mM borate buffer, pH 8.5, and immediately thereafter with 200 µL aliquots of toluene, followed by immediate vortex-mixing for 60 s and centrifugation (4000× g, 5 min, 18 °C). Aliquots (150 µL) of the upper organic phase were transferred into autosampler glass vials equipped with microinserts, and the samples were sealed and subjected to GC-MS analysis.

Procedure B. Solid amino acids were derivatized first with PFPA/EA (30 min, 65 °C) and then with 2 M HCl/CH₃OH or 2 M HCl/CD₃OD (30 min, 80 °C). Briefly, aliquots (100 µL) of a freshly prepared PFPA/EA solution were added, the glass vials were tightly sealed and heated for 30 min at 65 °C to prepare N-pentafluoropropionic amides of the methyl esters. After cooling the samples to room temperature, solvents and reagents were evaporated to dryness under a stream of nitrogen. Then, residues were reconstituted in 100 µL aliquots of a 2 M HCl/CH₃OH or 2 M HCl/CD₃OD solution and the glass vials were tightly sealed. Esterification was performed by heating the samples for 30 min at 80 °C. After cooling to room temperature, solvents and reagents were evaporated to dryness under a stream of nitrogen. Residues were treated directly with toluene (200 µL), shortly vortex-mixed, aliquots (150 µL) of the upper organic phase were transferred into autosampler glass vials equipped with microinserts, and the samples were sealed and subjected to GC-MS analysis.