Cto 20

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.

The HPLC analysis of jua extracts was performed according to previous studies [39 (link),40 (link)]. The HPLC apparatus (Shimadzu, LC20AT, Kyoto, Japan) consisted of an automated sample injector (SIL-20AC), controller (CBM-20), column oven (CTO-20), and diode array detector (SPD-M20A). A C18 stationary phase column (Shimadzu, Shim-Pack, VP-ODS-2, 25 × 0.6 cm, particle of 5 µm) was used. The mobile phase was composed of 0.1% (v/v) deionized water (A) and acetonitrile (B) in a proportion of 9.5 to 0.5 of A to B. The gradient elution followed the order: 5% B (10 min), 5–100% B (40 min), 100% B (5 min). The oven temperature was kept at 35 °C, and the elution rate was set at 1 mL/min during the analysis. The detection was performed between 190 and 400 nm. The coefficients of determination (R2) ranged from 0.9925 to 0.9999. The limits of detection (LOD) were in the range of 0.2 to 164.1 ng·mL⁻¹, while the limits of quantification (LOQ) ranged from 0.7 to 547.1 ng·mL⁻¹.