Ms 5973

The TMSO carbohydrate derivatives were analyzed in a gas chromatograph coupled to a quadrupole mass detector (Agilent GC-6890 and MS5973; Agilent, Palo Alto, CA, United States). Column, GC and MS conditions are described previously (Julio-Gonzalez et al., 2019 (link)). The identification of TMSO carbohydrate derivatives was carried out by the analysis of corresponding mass spectra and data of previously reported standards (Hernández-Hernández et al., 2011 (link)). These identifications were considered as tentative.

Ulvan was extracted from the residue following aqueous ethanol (75%) extraction with hot water (90 °C, 1 h × 3). Ulvan was recovered by ethanol precipitation and then freeze-dried. The ¹³C uptake in ulvan was measured by IR-MS. Semiquantitative ¹³C-NMR of ¹³C-labelled ulvan was performed by the inverse-gated decoupling method using JEOL NMR ECA600 (150 MHz)^{35 (link),36 (link)}. Ulvan was re-solubilised in D₂O-DMSO-d₆ (92:8) mixed solvent. NMR spectra were recorded at 40 °C with a repetition time of 10.69206 s. The chemical shift intensities were normalised to the DMSO-d₆ internal standard. The monosaccharide composition of ulvan was analysed by GC–MS (Agilent GC 8690 N and MS 5973; Agilent Technologies, CA, USA). Ulvan was hydrolysed into monosaccharides by aqueous trifluoro acetate (TFA; 2 M, 121 °C, 1 h). After removing TFA, the monosaccharides were derivatised to alditol acetate and analysed by GC–MS with a column of CBP-1 (Shimadzu GLC Ltd., Kyoto, Japan) operated at 140–160 °C (2 °C min⁻¹).

The determination of free terpenes and C₁₃-norisoprenoids was conducted by means of headspace solid phase microextraction (HS-SPME) in connection with gas chromatography and mass spectrometry (GC-MS), according to the method of Câmara et al. [26 (link)] and corresponding adaptations by Brandt et al. [27 ]. The following analytical equipment was applied: a multipurpose sampler MPS 2 (Gerstel GmbH & Co. KG, Mülheim an der Ruhr, Germany) was used for HS-SPME injection in combination with a GC-MS-System (GC 6890 and an MS 5973 (Agilent)). HS-SPME extraction was carried out with a 1 cm SPME fiber coated with 100 µm of polydimethylsiloxane (Supelco). Aroma compound separation was performed using a 30 m × 0.25 mm × 0.5 µm gas chromatography column (DB-Wax, J & W Scientific, Agilent) together with the mass spectrometer used in the SIM mode. For the instrumental control, acquisition and quantitative analysis of data, Agilent MassHunter workstation software was used. Examples of extracted ion chromatograms for TDN and linalool are provided in Supplementary Figure S1A,B.

A migration cell with 1 dm²‐migration area was obtained from Maeda Seisakusyo (Tokyo, Japan). The centrifugal condenser CVE‐100, Tokyo Rikakiki Co. (Tokyo, Japan) and the GC/MS system comprising GC 6890 and MS 5973 (Agilent Technologies, USA) were used.

Melting points were recorded on a hot-stage microscope (Reichert, Wien, Austria, Thermovar). Precoated silica gel PET foils (Sigma-Aldrich, St. Louis, MI, USA) were used for TLC analyses. GLC-FID analyses were performed on a Dani (Milan, Italy) GC 1000 chromatograph equipped with a PTV injector, using an Agilent (Santa Clara, USA) J&W DB-1 column (15 m × 0.25 mm × 0.25 μm) and recorded with a Dani DDS 1000 data station. GLC-MS analyses were recorded with an Agilent 6890N gas chromatograph interfaced with an Agilent MS5973 mass detector, using an Agilent J&W DB-5ms (30 m × 0.25 mm × 0.25 μm) column. Purifications by flash chromatography were performed using Merck 60 silica gel. ¹H-NMR and ¹³C-NMR spectra were recorded at 400 and 100 MHz, respectively, with a Jeol (Tokyo, Japan) 400 spectrometer referring chemical shifts to the residual solvent signal. The following notation was used to report NMR spectra: s = singlet, d = doublet, dd = double doublet, t = triplet, dt = double triplet, q = quadruplet. All the commercially available reagents and solvents were used as received.