Tracegold tg 5silms gc column

The synthesis procedures for HAs and production of 5-pentylisoxazol-3-ol are described in detail in the Supplementary Methods. The compounds were characterized by ¹H NMR and ¹³C NMR (400 MHz, AV400 spectrometer, Bruker). Exact masses of HHA, NHA, DHA, LHA, 2-octenoHA, 3-octenoHA, 2-octynoHA, 3-octynoHA and 7-octynoHA were determined by high-resolution mass spectrometry (HRMS) (electrospray ionization (ESI)-quadrupole-time-of-flight (QTOF) tandem mass spectrometry; maXis, Bruker Daltonics). Exact mass of 5-pentylisoxazol-3-ol was determined by gas chromatography-mass spectrometry (Trace 1310 GC with the Q Exactive GC, Thermo Scientific) using TraceGold TG-5Silms GC column (30 m length,  0.25 mm inner diameter, 0.25 µm film thickness, Thermo Scientific).

The chemical tissue analysis was performed as described in Strehse et al. (2017 (link)): Mussel tissues were thawed, whole meat was placed in a 50 mL polypropylene tube per mussel, and homogenized using a T25 Ultra-Turrax (Ika Works Inc., Staufen im Breisgau, Germany). Tissues were aliquoted into 1.0 g portions in 50 mL polypropylene tubes, and 5 mL gradient grade acetonitrile (Th. Geyer GmbH & Co. kg, Renningen, Germany) were added per tube. Each sample was mixed for one minute using a VF2 vortex mixer (Ika Works Inc., Staufen im Breisgau, Germany). Tubes were centrifuged for 5 min at 4 100 rpm (20 °C) with a Heraeus Megafuge 11R centrifuge (Thermo Fisher Scientific Inc., Waltham, MA, USA). Supernatants were decanted and filled with acetonitrile to a total volume of 10.0 mL. GC/MS-MS analysis was performed on a TSQ 8000 EVO Triple Quadrupole Mass Spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) with a Trace 1310 Gas Chromatograph (Thermo Fisher Scientific Inc., Waltham, MA, USA) and using a TraceGOLD™ TG-5SilMS GC column (30 m × 0.25 mm × 0.25 µm).