Precolumn

For qualitative and quantitative analysis of the prepared 5-KF and D-fructose solution, a Knauer Smartline HPLC-system (Knauer GmbH, Berlin, Germany) was used. The system was composed of a degasser (Knauer Smartline manager 5000), a pump (Knauer Smartline pump 1000), an autosampler (Knauer Smartline autosampler 3800), a column oven (Knauer Column-Thermostat Jetstream 2 Plus), an RI detector (AZURA RID 2.1L), and an ultraviolet (UV) detector (Knauer Smartline UV detector 2600), which measured the absorbance at a wavelength of 210 nm. Sample separation was achieved using the Eurokat H column (300 × 8 mm; Knauer GmbH) and a precolumn (30 × 8 mm; Knauer GmbH) heated to 65^°C. The mobile phase, 5 mM H₂SO₄, was applied at a flow rate of 0.6 mL min^–1. The injection volume was set to 20 μL. Data evaluation and control of the HPLC system were accomplished using ClarityChrom^® 8.2.3 (Knauer GmbH). Peak assignment and quantification were performed by applying external standards of D-fructose and 5-KF in concentrations of 0.5, 1, and 2 mM.

LC-MS analysis was performed on a Shimadzu HPLC system (controller CBM-20A, two pumps LC-20 AD, a column oven CTO-20 AC and a photo diode array detector SPD-M20A; Shimadzu, Darmstadt, Germany) coupled to a Triple Tof 4600 mass spectrometer (AB Sciex, Canby, USA). The separation of extracted compounds was realised on a Knauer Vertex Plus column (250 × 4 mm, 5 μm particle size, packing material ProntoSIL 120–5 C18-H) with precolumn (Knauer, Berlin, Germany). The column oven temperature was set to 30 °C and 25 μl of undiluted methanolic seagrass extract prepared as described above was injected. The solvent flow rate was 0.8 ml/min. In this time, a gradient was run from 10 to 90% B from minute 0 to 35, 2 min of 90% B, switch to 10% B in 1 min and subsequent equilibration at 10% B for 2 min. Solvent A (water) and B (methanol) were both supplemented with 2 mM ammonium acetate and 0.01% acetic acid. Mass spectra were monitored between 100 and 800 Da in negative ionisation mode. In addition, MS/MS spectra were generated with a collision energy of − 30 eV and measured between 50 and 800 Da. Spectra for the most prominent peaks were compared to database entries in MassBank [35 (link)] and ReSpect [36 (link)] for identification.

For LC-MS analysis, an HPLC (Shimadzu, Darmstadt, Germany) coupled to a triple TOF mass spectrometer (AB Sciex, Canby, USA) was used. Water (A) and methanol (B) supplemented with 2 mM of ammonium acetate and 0.01% acetic acid were used as solvents. The flow rate was 0.8 mL/min, and a gradient was applied with 10% to 90% B over 35 min, two min of 90% B, and switching to 10% B in one min, followed by two min of 10% B. The column oven temperature was 30 °C, and 10 µL of the sample was injected into a Knauer Vertex Plus column (250 × 4 mm, 5 µm particle size, packing material ProntoSIL 120-5 C18-H) with precolumn (Knauer, Berlin, Germany). The ion source was set to negative electrospray ionization mode (ESI–) and mass spectra were recorded between 100–1000 Da. MS/MS spectra were measured at a collision energy of −30 eV between 50–800 Da. Precursor masses and MS/MS peaks were compared to database entries in MassBank [60 (link)] and ReSpect [61 (link)] for identification.