Ulc ms grade

Polar metabolites were extracted from the nuclei pellet with the addition of 300 μl of ice-cold methanol (ULC/MS grade, Biosolve, 136841) supplemented with 10 μl of adonitol (50 μm/ml; Alfa Aesar, L03253.06) as an internal standard and incubation for 15 min at 72°C. The methanol/nuclei suspension was further mixed with 300 μl of ice-cold MilliQ H₂O and centrifuged at 15,000 rpm at 4°C for 10 min. The supernatants were transferred into amber glass vials (Agilent, 5183-2073), dried with the Genevac EZ-2 Plus evaporator (program, hplc fraction; temperature, 30°C), and stored at −80°C until analysis with GC-MS or LC-MS.
For the extraction of polar metabolites from whole-cell lysates, 300 μl of whole-cell lysates in incubation buffer was mixed with 600 μl of ice-cold methanol supplemented with 10 μl of adonitol (50 μm/ml) and was incubated for 15 min at 72°C. Ice-cold MilliQ H₂O (600 μl) was added to the methanol/whole-cell lysate mixture, and the rest of the steps were performed as indicated for the nuclei experiments.

Organic solvents were ULC-MS grade and purchased from Biosolve (Valkenswaard, The Netherlands). Chemicals and standards were analytical grade and purchased from Sigma-Aldrich (Zwijndrecht, The Netherlands). Water was obtained on the day of use from a Milli Q instrument (Merck Millipore, Amsterdam, The Netherlands).

UPLC–MS analysis of the crude extracts (concentration 1 mg/mL) were performed on an Acquity UPLC I-Class System coupled to a Xevo G2-XS QToF Mass Spectrometer (Waters, Milford, MA, United States) controlled by MassLynx version 4.1. Samples were injected and separated on an Acquity UPLC HSS T3 column (High Strength Silica C18, 1.8 mm, 100 × 2.1 mm, Waters, Milford, MA, United States) at a temperature of 40 °C with an injection volume of 0.2 µL. A binary mobile phase system comprised of mobile phase A: 99.9% MilliQ®-water / 0.1% formic acid (ULC/MS grade) and mobile phase B: 99.9% acetonitrile (MeCN, ULC/MS grade, Biosolve BV, Dieuze, France)/0.1% formic acid. They were pumped at a rate of 0.6 mL/min with a linear gradient starting with 99% A from minute 0–11.5, followed by 0% A for 1 min (11.5–12.5), and back to the starting condition for 2.5 minutes. MS was done with an electrospray ionization source over a mass range of m/z 50–1600 Da in the positive mode with a capillary voltage of 0.8 kV, cone gas flow of 50 L/h, desolvation gas flow of 1200 L/h, source temperature of 150 °C, desolvation temperature of 550 °C with sampling cone and source offset at 40 and 80, respectively. The MS/MS experiments were carried out in tandem with ramp collision energy (CE): Low CE from 6 to 60 eV and a high CE of 9 to 80 eV. Solvents and PDA medium extracts were also analyzed.

Doxapram (C₂₄H₃₀N₂O₂, 97.7%, 378.2 g/mol) for method validation was bought from Biozol Diagnostika Vertrieb (Eching, Germany). 2-Ketodoxapram (C₂₄H₂₈N₂O_3, 98.8%; 392.2 g/mol); the stable isotopically labelled internal standards (IS) doxapram-d5 (99.6%, 383.2 g/mol) and 2-ketodoxapram-d5 (99.6%, 397.2 g/mol) were synthesised by TLC Pharmaceutical Standards (Newmarket, ON, Canada).
Acetonitrile (ACN) and formic acid (FA) were purchased from Biosolve (ULC/MS grade; Valkenswaard, The Netherlands), and tert-butyl methyl ether (TBME), boric acid, sodium hydroxide, and hydrochloric acid were purchased from Merck (Darmstadt, Germany). Ultrapure water was freshly prepared with an arium^® mini system (Sartorius, Göttingen, Germany). Analyte-free porcine plasma and brain tissue for assay validation was available from untreated control animals from previously performed studies.

Benzophenone-3 (BP3; 98%, CAS No. 131-57-7) was obtained from Sigma-Aldrich (Taufkirchen, Germany). Bis(tri-n-butyltin) oxide (TBTO; 97%, CAS No. 56-35-9, abcr GmbH, Karlsruhe, Germany) was used as positive control. Ethanol (EtOH; ≥99.8%, Carl Roth GmbH & Co. KG, Karlsruhe, Germany) was used as solvent to spike BP3 in water samples for matrix-matched calibrations. Tetrachloroethylene (TCE; HPLC grade, ≥99.9%, Sigma-Aldrich, Taufkirchen, Germany) was used as extraction solvent and formic acid (FA; Biosolve BV, Valkenwaard, The Netherlands) for adjusting the pH of water samples. For the analytical system, acetonitrile (ACN; ULC/MS grade, ≥99.99%; Biosolve BV, Valkenwaard, The Netherlands), and MilliQ water (ultrapure water purification system arium 611DI, Sartorius AG, Göttingen, Germany), both containing 0.01% FA, were used. A synthetic salt mix (Pro-Reef salt, Tropic Marin, Prof. Dr. Biener GmbH, Wartenberg, Germany) was used for the preparation of artificial seawater. Further details on physicochemical properties [69 ,70 ,71 ] of the active ingredients (BP3, TBTO), and chemicals used in additional bioassays can be found in Tables S1 and S22.