Ods 3 c18

ATP-related compounds were determined proposed by Wang et al. [83 (link)] with a reversed-phase high performance liquid chromatogram (RP-HPLC) method (Waters 2695, Milford, CT, USA). Sample preparation method was as follows: 5 g minced sea bass muscle homogenized with 10 mL10% perchloric acid (PCA) and centrifuged at 8000 g for 15 min at 4 °C. The precipitate was stirred with 10 mL 5% PCA and centrifuged at 8000 g for 10 min at 4 °C twice. The supernatant pH was adjusted to 6.5 after the supernatant was merged and added with 15 mL of distilled water. After 30 min, take the supernatant fixed in a 50 mL volumetric bottle with ultrapure water. Finally, the supernatant was filtered with a 0.22 µm membrane and applied to the RP-HPLC procedure. The chromatographic conditions were as follows: column: Shimadzu ODS–3 C18 (4.6 mm × 250 mm, 5 mm); mobile phase: A–20 mmol/L KH₂PO₄:20 mmol/L K₂HPO₄ (v/v 1:1), adjusted to pH 6.5 with phosphoric acid; B–Methanol; column temperature: 30; injection volume: 10 mL; detection wavelength: 254 nm; flow rate: 1.0 mL/min; gradient: 0–6 min 100%A, 6–15 min B increases linearly to 8%, 15–20 min B increases linearly to 35%, 20–22 min 35%B,22–24 min B decreases linearly to 0%, 24–30 min 100% A. The K value was calculated as follows: $K\mathrm{value}(\%)=\frac{\mathrm{HxR}+\mathrm{Hx}}{\mathrm{ATP}+\mathrm{ADP}+\mathrm{AMP}+\mathrm{IMP}+\mathrm{HxR}+\mathrm{Hx}}\times 100$

The experiments were performed using a LC-20A (SHIMADZU, Japan) High-Performance Liquid Chromatograph and a SHIMADZU ODS3-C₁₈ chromatographic column (4.6 mm × 250 mm, 5 µm). The mobile phase consisted of acetonitrile as Solvent A and 1% formic acid as Solvent B at a flow rate of 1 mL/min. The column temperature was maintained at 30 °C, and the injection volume was 20 µL.
The gradient elution program of the standard solution included 35–55% A at 0–10 min; 55–75% A at 10–30 min; 75% A at 30–31 min. The detection wavelength was 254 nm.
The gradient elution program of the transformation assay in vitro included 20–50% A at 0–10 min and 50–90% A at 10–35 min. The detection wavelength was 280 nm.
The gradient elution program of the transformation assay in vivo included 35–55% A at 0–10 min; 55–75% A at 10–30 min; 75–95% A at 30–40 min. The detection wavelength was 254 nm.

Solubility was determined on the samples obtained as follows. Samples were made into a saturated solution and filtered through a filter paper (0.22 µm). The filtrate solution was analyzed by HPLC (Agilent G7129, San Diego, CA) equipped with column Shimadzu ODS-3 C₁₈ (Shimadzu, Kyoto, Japan) (4.6 × 250 mm, 5 μm) at 30 °C and diode-array detector was set to a wavelength of 218 nm. The eluent was acetonitrile-deionized water (40:60, v/v), with the flow rate of 1.0 mL/min.