Uhplc q orbitrap

For each sponge specimen (P. ficiformisn = 8, C. reniformisn = 10, C. cramben = 9, C. nuculan = 10), ca. 500 mg of freeze-dried material was crushed and sonicated with 4.5 mL methanol (MeOH) for 30 s. Then, 15 mL of methyl tert-butyl ether (MTBE) were added and left 1 h at room temperature under shaking. Upon addition of 3.75 mL of chromatography water, the sample was mixed and then centrifuged to induce phase separation^{133 (link)}. The upper organic phase was recovered, dried and kept in glass vials at -20 °C until use. Dry extracts were resuspended and injected in a Thermo Scientific Dionex UltiMate 3000 chromatograph coupled to a Thermo Scientific Q Exactive focus quadrupole-Orbitrap mass spectrometer (UHPLC-q-Orbitrap, Thermo Fisher Scientific, MA, USA) operated in full scan-data dependent MS2 (Full MS-ddMS2) discovery acquisition mode. Further details about UHPLC-q-Orbitrap configuration are reported in section 1.4 of the SM.

Most of the chemicals were purchased from Aladdin, energy-chemical, TCI, or Alfa Aesar. NMR spectra were recorded on Bruker Avance 600 spectrometers. Chemical shifts (ppm) were given relative to the solvent. Melting points of the target molecules were determined using a Shanghai Yice WRX-4 melting point meter. High-resolution mass spectrometry data were determined on a Thermo Scientific (UHPLC-Q-Orbitrap).

The analysis of neurotransmitters, including histamine, L-histidine, L-glutamic acid, gamma-aminobutyric acid (GABA), acetylcholine, L-DOPA, 3-methoxytyramine, serotonin, 5-hydroxyindole-3-acetic acid, glycine, L-glutamine, L-valine, 3-hydroxytyramine and choline, was performed with a method developed in a previous study⁹³ . Briefly, per sample, 18 zebrafish larvae were pooled and each sample was spiked with 100 ppb isotope-labeled internal standards and then homogenized. The homogenized tissue samples were extracted with 200 μL of 80:20 v/v ACN/Milli-Q water containing 0.1% formic acid and 1% ascorbic acid and 200 μL ACN. After protein precipitation, the supernatants were dried and redissolved in 100 μL of Milli-Q water containing 0.1% formic acid for UHPLC-MS/MS analysis. Neurotransmitters were quantified by a high-performance liquid chromatography system coupled to a Q-Exactive plus quadrupole-Orbitrap mass spectrometer (UHPLC-Q-Orbitrap, Thermo fisher, US). The separation of neurotransmitters was carried out with an Acquity UPLC HSS T3 column with Milli-Q water containing 0.1% formic acid and ACN containing 0.1% formic acid as mobile phase. The MS acquisition was performed in the full scan mode and parallel reaction monitoring mode. The experiment was performed once (n = 4 per treatment group).