Acetanilide

For preparation of the ESI and SESI buffer solution, water (H₂O, Optima, Fisher Chemical, LC/MS grade), methanol (MeOH, Optima, Fisher Chemical, LC/MS grade) and formic acid (FA, Merck, for analysis, purity 98–100%) were used. As model compounds, six readily available isobars, which are separable in MS¹ but co‐fragment in MS², were selected and are shown in Figure 2. Namely, benzothiazole (1, TCI, purity >96.0%), pyridine‐2,6‐dicarbaldehyde (2, Fluorochem, purity ≥98%), 3H‐pyrrolo[2,3‐d]pyrimidin‐4(7H)‐one (3, Fluorochem, purity ≥95%), adenine (4, Sigma‐Aldrich, purity ≥99%), acetanilide (5, Sigma‐Aldrich, purity ≥99.5%) and N,N‐dimethylbenzylamine (6, Sigma‐Aldrich, purity ≥99%) were used. For an exemplary application, azelaic acid (7, Sigma‐Aldrich, purity ≥98%) and 10‐hydroxydecanoic acid (8, Apollo Scientific, purity ≥95%), which were previously identified in a breath metabolomic study,³⁸ were used for control measurements. Commercial calibration solutions (Pierce ESI Positive Ion Calibration Solution and Pierce ESI Negative Ion Calibration Solution, Thermo Scientific) were used to mass calibrate the instrument with the ESI source.

Acetanilide and substituted anilines were purchased from Sigma-Aldrich and were used without further purification. Other Acetanilide derivatives (Table 1) were synthesized from the respective commercially available substituted aniline precursors following literature procedure.²⁰ Acetic anhydride (12.7 mmol) was added to the aniline precursors (10 mmol) suspended in water (3 mL). The mixture was vigorously stirred in a warm water bath. Once the solid particles got dissolved, the reaction mixture was cooled. The solid product was then filtered and washed with cold water. The solid acetyl derivatives were finally recrystallized from hot water and dried at ambient conditions. All the synthesized compounds were characterized using ¹H and ¹³C 1D NMR spectra acquired in DMSO-d₆ as the solvent.

Monosodium phosphate (NaH₂PO₄), disodium phosphate (Na₂HPO₄), ammonium bicarbonate (NH₄HCO₃), trimethylsilyl
propionic acid-d₄ sodium salt (TSP), acetanilide, N-ethylmaleimide (NEM), GSH (reduced, GSH), Cys, hydrochloric
acid, and sodium hydroxide were obtained from Sigma-Aldrich (St. Louis,
MO) or Fisher (Waltham, MA). Deuterium oxide (D₂O) was
procured from Cambridge Isotope Laboratory (Tewksbury, MA). Deionized
(DI) water was purified using an in-house Synergy Ultrapure Water
System from Millipore (Billerica, MA). All chemicals and solvents
were used without further purification.

Methanol (MeOH) and acetonitrile (MeCN) (LC-MS grade) were obtained from Fisher Scientific (Loughborough, UK). Formic acid (99.5+) was obtained from Merck KGaA (Darmstadt, Germany). Ultra-pure water was prepared by an in-house water purification system (Milli-Q, Integral 3, Millipore Co., USA). Ultima Gold liquid scintillation counting (LSC)-cocktail was obtained from PerkinElmer (Waltham, MA, USA). All other chemicals and solvents were of the highest quality commercially available.
Specific chemicals used for analysis by accelerator mass spectrometry (AMS) included Australian National University sucrose with a certified ¹²C/¹⁴C ratio purchased from the National Institute of Standards and Technology (Gaithersburg, MD, USA), and acetanilide and paracetamol from Sigma-Aldrich (St. Louis, MO, USA).