Acetonitrile acn

Dried HPLC fractions were dissolved in loading buffer (LB1X: 2% SDS BIORAD, 50mM TRIS-HClpH6.8, 10% Glycerol SIGMA, and bromophenol blue BIORAD), fractionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained with GelCode™ Blue Safe Protein Stain (Thermo Fisher Scientific). After destaining, two bands were cut from lanes 22 and 26, respectively. The bands were in situ hydrolyzed by trypsin as reported in [39 (link)]. Briefly, gel bands were further destained alternating washes with acetonitrile (ACN) (Honeywell, Charlotte, NC, USA), 50 mM ammonium bicarbonate (NH4HCO3) (Sigma, St. Louis, MO, USA), and cysteine residues reduced by 10 mM of dithiothreitol (Sigma, St. Louis, MO, USA), and then alkylated in 55 mM iodoacetamide (Sigma, St. Louis, MO, USA). Following extensive washings to remove the excess reagents, gel bands were then treated with trypsin. Peptide mixtures were extracted in 0.2% HCOOH and ACN and vacuum dried by a Savant SpeedVac System (Thermo Fisher Scientific, Waltham, MA, USA).

Acetonitrile (ACN) and methanol were purchased from Honeywell (Muskegon, MI, USA). Distilled water (18.2 MΩ) was purified using a Milli-Q system (Millipore, Billerica, USA). Leucine enkephalin was acquired from Sigma-Aldrich (St. Louis, MO, USA). Formic acid (HPLC grade) was purchased from J&K Chemical Ltd (Beijing, China). All other chemicals were acquired from Sigma-Aldrich (St Louis, MO, USA) unless otherwise specified.

Analytical standards of all analytes were purchased from Sigma Aldrich (Steinheim, Germany). Isotopically labeled internal standards (IS) were obtained as follows: PA-d4, NA-d4, and 3-IAA-d2 from Merck (Darmstadt, Germany); KA-d5 and Trp-indole-d5 from CDN isotopes (Pointe Claire, QC, Canada); XA-d4 from Santa Cruz Biotechnology (Dallas, TX, USA); 3-OH KYN-d3 from Buchem BV (Minden, Apeldoorn, Netherlands); QA-d3 and 5OH IAA-d6 from MedChemTronica (Sollentuna, Sweden); 3-OH AA-d3 and I3LA-d5 from LGC Standards (Łomianki, Poland); and AA-ring-13C6 from Eurisotope (Saint-Aubin, France). Sodium carbonate (Na₂CO₃), derivatization reagent (BNAP), ascorbic acid (AsA), and formic acid (FA) for LC-MS were obtained from Sigma Aldrich. LC-MS grade methanol (MeOH) and acetonitrile (ACN) were acquired from Honeywell (Seelze, Germany). Ultra-pure water (MPW) was made from distilled water using the Merck Millipore purification system from Merck.

Chemicals and solvents (analytical grade)
were purchased from the following sources: α-cyano-4-hydroxycinnamic
acid 98% (CHCA) (Sigma-Aldrich), 2,5-dihydroxybenzoic acid 98% (DHB)
(Sigma-Aldrich), norharmane 98% (Nor) (Acros Organics), 1,5-diaminonaphthalene
97% (DAN) (Sigma-Aldrich), 2′,4′,6′-trihydroxyacetophenone
99.5% (THAP) (Sigma-Aldrich), 2,6-dihydroxyacetophenone 99.5% (DHAP)
(Sigma-Aldrich), acetonitrile (ACN) (Honeywell), chloroform (Acros
Organics), methanol (Carl Roth), ammonium acetate (AmAc) (VWR). ammonium
sulfate (AmS) (Sigma-Aldrich), heptafluorobutyric acid (HFBA) (Sigma-Aldrich),
and trifluoroacetic acid (TFA) (Sigma-Aldrich). All chemicals used
in this study were stored, handled, and disposed of according to good
laboratory practices (GLP).

Dexamethasone (purity ≥ 97%) and levofloxacin (purity ≥ 98%) were obtained from Sigma Aldrich (St. Louis, MO, USA) and BI 113823, as well as BI 1026706 from the in-house dispensary at Boehringer Ingelheim (BI). Melanin from Sepia officinalis and synthetic melanin were obtained from Sigma Aldrich together with sodium hydroxide (NaOH), bovine serum albumin (BSA) and formalin solution (10%, neutral buffered). Dimethyl sulfoxide (DMSO) was purchased from Merck Millipore (Burlington, MA, USA). Phosphate buffered saline (PBS) was made from one vial of phosphate buffer powder (pH 6.6, Sigma Aldrich) and 34.2 g of NaCl (Sigma Aldrich) by mixing both in purified water (Milli-Q, MilliporeSigma, Burlington, MA, USA) in a total volume of 3.8 L. The pH was adjusted to 6.5 with HCl. Ethanol (EtOH) was obtained from Carl Roth GmbH (Karlsruhe, Germany). Acetonitrile (ACN) was purchased from Honeywell (Charlotte, NC, USA). Tween-80 (Polysorbat 80) and hydroxyethyl cellulose (Natrosol) for the oral suspension formulation of the drugs were obtained from Dr. W. Kolb AG (Hedingen, Switzerland) and Ashland Industries Deutschland (Düsseldorf, Germany), respectively.

DON was obtained from Romer Labs (Tulln, Austria) and CER was purified as previously described [28 (link)]. Cell culture media and supplements were obtained from Gibco® Life Technologies (Karlsruhe, Germany). KH₂PO₄, KCl, NaCl, Na₂HPO₄, D-glucose, HEPES, CaCl₂, MgCl₂, glycin, formaldehyde, sodium dodecyl sulfate (SDS), NaOH and Triton X-100 were purchased from Carl Roth (Karlsruhe, Germany) and Lucifer Yellow CH di-lithium salt from Santa Cruz Technologies (Dallas, TX, USA). For LC-MS/MS measurements, MS grade water was purchased from VWR (Fontenay-sous-Bois, France), acetonitrile (ACN) and methanol (MeOH) from Honeywell (Seelze, Germany). Acetic acid was obtained from Merck (Darmstadt, Germany) and ammonium acetate (both LC-MS grade) from Sigma Aldrich (Vienna, Austria). DON-3-sulfate, DON-15-sulfate and DON-3-glucuronide were kindly provided by Dr. Philipp Fruhmann (Technical University of Vienna) and synthesized as previously described [44 (link)].