Inertsil ods 3 c18 column

The 3,5-dinitrosalicylic acid (DNS) method was used to determine the reducing sugar content. The ethanol content was assayed using gas chromatography (GC) as described by Liang et al. [19 (link)] with slight modifications. The sample was analyzed on a GC system (Agilent Technologies, Inc., Palo Alto, CA, USA), and a DB-Wax column (30 m × 0.25 mm, 0.25 μm film thickness, Agilent Technologies, Inc., Palo Alto, CA, USA) was used, with column and injector temperatures of 60 °C and 200 °C, respectively.
The GABA content was quantified using high-performance liquid chromatography (HPLC) (Waters 2695, Waters corp., Reno, NA, USA) as previously described [14 (link)]. The separation column was an Inertsil ODS−3 C18 column (4.6 mm × 150 mm, 5 μm, Shimadzu, Japan). The mobile phases used were A (0.05 M sodium acetate: methanol: tetrahydrofuran, 84: 15: 1, V/V/V) and B (methanol). The flow rate and temperature of the elution phase were maintained at 1.0 mL/min at 30 °C.
The organic acid analyses were also performed using HPLC as previously described [14 (link)]. The separation column was an ionic exchange resin Bio-Rad Aminex HPX−87H column (7.8 mm × 300 mm, 9 μm; Bio-Rad Laboratories, Inc., Hercules, CA, USA) with a flow rate of 0.6 mL/min and 5 mmol/L sulfuric acid as the mobile phase. The UV detection wavelength was 210 nm, and the column oven temperature was maintained at 60 °C.

SXNI was analysed through liquid chromatography which was performed on a Shimadzu inertsil ODS-3 C18 column (5 µm, 250 mm × 4.6 mm), at a flow rate of 1 mL/min using high-performance liquid chromatography (HPLC) on a Waters 2695 system (Waters, USA), and the detection wavelength was 360 nm. The mobile phase A was acetonitrile, and B was water containing 0.4% phosphoric acid. The gradient profile for the LC pumps under the final chromatography conditions are illustrated in Table 1.

Analysis for metabolites was carried out using a Shimadzu HPLC equipped with an SPD-M20A photodiode array detector and a supplementary L-20A autosampler and managed by a LabSolutions LC Workstation (Shimadzu). The analytes were separated on an Inertsil ODS-3 C₁₈ column (5.0 μm, 4.6 mm by 250 mm; Shimadzu) operating at 30°C, and compounds were detected at 280 nm. The mobile phase solvents A and B were water and acetonitrile, both containing 0.05% phosphoric acid. Gradient elution at a flow rate of 0.8 ml/min was performed as follows: 0 to 25 min, 85% to 30% A; 25 to 30 min, 30% to 5% A; 30 to 31 min, 5% to 85% A; 31 to 35 min, 85% A. High-resolution mass spectrometry and tandem mass spectrometry were conducted on a Thermo Q Exactive Plus. ¹H and ¹³C NMR was conducted on a Varian Mercury Plus 400 NMR spectrometer. The compounds 5, 7a, and 7b were characterized in our previous work (15 (link)).