Sulfadimethoxine

LC-MS-grade water and acetonitrile (ACN) were purchased from Fisher Scientific (Houston, TX, USA). Dansyl chloride (DC), n-butanol, acetone, d₅-tryptophan, tripentadecanoin, sulfadimethoxine, and amino acid standards were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Amoxicillin, azithromycin, cefuroxime, chloramphenicol, chlortetracycline, ciprofloxacin, clarithromycin, colistin, danafloxacin, decoquinate, dexamethasone, diclofenac, difloxacin, doxycycline, enrofloxacin, erythromycin, florfenicol, flumethasone, griseofulvin, ibuprofen, levofloxacin, lincomycin, maduramicin, mefenamic acid, monesin, narasin, nicarbazin, norfloxacin, oxytetracycline, paracetamol, propranolol, robenidine, sarafloxacin, salinomycin, spectinomycin, sulfachloropyridine, sulfadiazine, sulfadimethoxine, sulfamerazine, sulfamethasone, sulfamethoxazole, sulfamethoxypyridazine, sulfapyridine, sulfaquinoxaline, sulfathiazole, tetracycline, trimethoprim, and tylosin with a purity above 98% were bought from Sigma-Aldrich (St. Louis, MO, USA). Anhydrous citric acid, trichloroacetic acid (TCA), ethylenediaminetetraacetic acid disodium salt (EDTA), and disodium hydrogen phosphate dehydrate were also purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile (ACN), methanol (MeOH) (HPLC grade ≥ 99%), and formic acid (purity > 99% for analysis) were obtained from Acros Organics (Geel, Belgium). Purified water, with a resistivity higher than 18.0 MU, was prepared in the laboratory with a Milli-Q system from Millipore (Burlington, MA, USA).

Dried tissue samples were resuspended in 50% methanol (Fisher Optima; LC-MS grade) spiked with 2 μM sulfadimethoxine (Sigma-Aldrich) as internal control (except for heart samples, which were resuspended in 50% methanol), pooling aqueous and organic extracts together. Dried plasma samples were resuspended in 150 μl of HPLC-grade water spiked with 0.5 μM sulfadimethoxine as internal control. LC was performed using a Thermo Scientific Vanquish UHPLC system fitted with 1.7 μm 100 Å Kinetex C8 column (50 × 2.1 mm) (Phenomenex; GI tract and heart) or 1.6 μm 100 Å Luna Omega Polar C18 column (50 × 2.1 mm) (Phenomenex; plasma samples and cell-culture samples), using water with 0.1% formic acid as mobile phase A and acetonitrile with 0.1% formic acid as mobile phase B. Data-dependent MS/MS experiments were performed on a Q Exactive Plus (Thermo Scientific) high-resolution MS, under the control of Xcalibur and Tune software (Thermo Scientific). Ions were generated for MS/MS analysis in both positive and negative ion mode using heated electrospray ionization source. Calibration of the instrument was performed using recommended commercial calmix from Thermo Scientific. See table S2 for detailed instrumental parameters.