2690 separation module

HPLC analysis was performed using an HPLC system (Waters, Milford, MA, USA) equipped with a 2690 separation module and Waters 996 DAD with an ZORBAX Eclipse XDB-C18 column (250 mm × 4.6 mm, 5 μm; Agilent Technologies, Inc., Santa Clara, CA, USA). The mobile phase consisted of 0.1% formic acid in ionized water (solvent A) and 0.1% formic acid in methanol (solvent B). The ratio of the mobile phase was maintained at A:B 95:5 (0–5 min), 85:15 (5–10 min), 45:55 (10–25 min), and 95:10 (25–40 min) at a flow rate of 0.8 mL/min. UV–Vis absorption spectra were recorded from 200–400 nm during the HPLC analysis and the quantification of individual compounds were based on peak areas at 220 nm.

The fermented T. japonica fruit extract (FTJ) was sequentially fractionated with hexane, chloroform, ethyl acetate, butanol, and water. The water fraction was concentrated using a rotary vacuum concentrator (RE100-pro; DLAB Scientific Inc., USA). Six fractions were separated using a Sephadex-LH20 column and a RP silica column in sequence and were purified via high-performance liquid chromatography (HPLC). HPLC was performed using a Waters 2690 Separation Module and a Waters 2487 Dual λ Absorbance Detector. The analytical column (Xterra C18 ODS, 0.5 × 250 nm) was packed with LiChrospher 100RP-18 (15 μm; Merck Co.). The final compound obtained via a further HPLC process was confirmed to be a tripeptide by nuclear magnetic resonance (NMR; 1H, 13C, 2D-NMR).

The culture supernatants were analyzed by HPLC for maltose, glucose and ethanol using a Waters 2690 Separation Module and Waters System Interphase Module liquid chromatography coupled with a Waters 2414 differential refractometer and a Waters 2487 dual λ absorbance detector (Waters, Milford, MA). A Fast Juice Column (50 × 7.8 mm, Phenomenex, Torrance, CA) and a Fast Acid Analysis Column (100  × 7.8 mm, Bio-Rad, Hercules, CA) and an Aminex HPX-87H Organic Acid Analysis Column (300  × 7.8 mm, Bio-Rad) were equilibrated with 2.5 mM H₂SO₄ in water at 60°C and samples were eluted with 2.5 mM H₂SO₄ in water at a 0.5 ml/min flow rate. Data were acquired with Waters Millennium software.

Extracts were prepared by dissolving 0.05 g of the freeze-dried powdered samples of watermelon using 10 mL HPLC grade water in a 15 mL conical plastic tube. The solution was kept in an automatic shaker for 30 min, centrifuged, and then the supernatant was filtered using a 0.45 μm microsyringe filter. Extract solutions were stored at −20 °C for subsequent analysis.
The analysis of the citrulline and arginine was conducted using a Waters HPLC system equipped with a 2690 separation module and waters 996 diode array detector (Milford, MA, USA). UV-Vis detection was set at 200 nm, and the column temperature was held at room temperature. Separation of the compounds was done using a Gemini C₁₈ (3 µm partial size, 250 × 4.6 mm, Phenomenex, Torrance, CA, USA) column. The mobile phase was 0.1% phosphoric acid in an aqueous solution, and the elution mode was isocratic. Flow rate and total run time were set at 0.3 mL/min and 15 min, respectively. Extraction and analysis of all the samples were carried out in triplicates. The citrulline and arginine concentrations were calculated using linear calibration functions prepared using the serial dilutions (0.1, 1, 10, 25, 50, 100, 250, 500, and 1000 μg/mL) of commercial standards, and the final results were expressed as milligram per gram (mg/g) of freeze-dried watermelon sample.