W2690 5

Using a high‐performance liquid chromatography (HPLC) (W2690/5, Waters, USA) equipped with a diode array detector (DAD) (Waters, USA) and a C18 column (5μm, 4.6 × 250 mm; Shanghai ANPEL Science Instrument Co., Ltd, Shanghai, China) with a mobile phase, containing dichloromethane:acetonitrile:methanol (20:70:10, v/v/v) under isocratic condition with a flow rate of 1.0 ml/min to analyze astaxanthin, this method was determined by Sun et al. (2018) with slight modifications. Briefly, 0.5 g of krill oil mixed with 5 ml dichloromethane and then added 0.1 mol/L sodium hydroxide (NaOH) in methanol at 4°C for 9 hr in darkness for complete saponification. The determined wavelengths were 476 nm for astaxanthin and were identified and quantified by comparing with the standards (Shanghai Macklin Biochemical Co., Ltd, Shanghai, China).

VST concentration was determined using HPLC. The HPLC system included a pump (W2690/5; Waters Corporation, Milford, MA, USA), ultraviolet detector (W2489; Waters Corporation), data station (Empower 3; Waters Corporation), and chromatographic C18 column (250 × 4.6 mm, 5 μm; Shiseido, Tokyo, Japan), maintaining a flow rate of 1.0 mL per minute at 25 °C. The isocratic mobile phase was composed of acetonitrile and distilled water (60:40 [v/v]). The pH was adjusted to 3.0 using 10% phosphoric acid. Finally, 20 μL of each sample was injected into the column, and the absorbance was measured with ultraviolet detection at 247 nm.

HPLC analysis was performed to determine the concentration of VST. The HPLC system consisted of a pump (W2690/5; Waters Corporation, Milford, MA, USA), an ultraviolet detector (W2489; Waters Corporation, Milford, MA, USA), and a data station (Empower 3; Waters Corporation, Milford, MA, USA). Using a C18 column (250 × 4.6 mm, 5 μm; Shiseido, Tokyo, Japan), the isocratic mobile phase, composed of distilled water and acetonitrile (40:60 [v/v]) and pH-adjusted to 3.0 by adding 10% phosphoric acid, was eluted at a flow rate of 1.0 mL/min at 25 °C. Finally, aliquots (20 μL) of each sample were injected into the column, and the absorbance was measured with ultraviolet detection at 247 nm. The calibration curve was obtained by least-square linear regression in the range 10–120 μg/mL, with a coefficient determination (R²) value of >0.99.

ATP‐related compounds were determined based on previous procedures described by Yokoyama et al. (Yokoyama et al., 1992). The 5 g of fish fillets was accurately weighed before adding 10 ml of cold perchloric acid (10%, v/v), and then centrifuged (H2050R, Xiangyi Co., Ltd.) at 10,000 rpm at 4°C for 15 min after homogenization for 2 min. The same treatment process was repeated twice except for adding 5 ml of cold perchloric acid (5%, v/v).
The collected supernatants were combined and adjusted to pH 6.50 with 1 mol/L KOH, and then stood for 30 min. The supernatants were diluted to 50 ml, shaken and filtered through a 0.22 μm membrane. All operating conditions were conducted at 4°C.
Amounts of ATPs were determined by high‐performance liquid chromatography (HPLC) (W2690/5, Waters Co., Ltd) according to previous report (Qiu et al., 2015). ODS‐SPC¹⁸ column (4.6 mm × 250 mm, 5 μm) was employed for the separation of ATPs at 28°C. The mobile phase A and B were 0.05 mol/L KH₂PO₄ and K₂HPO₄ (1:1, v:v), and methanol, respectively. To improve the separation efficiency of ATPs, the flow rate and detection wavelength was set as 1.0 ml/min and 254 nm, respectively.

Biogenic amines were extracted and analyzed according to Özogul, Taylor, Quantick, and Özogul (2002) using an HPLC apparatus (W2690/5, Waters) and SUPELCOSIL LC‐18‐T, 250 × 4.6 mm, 5 µm column. All biogenic amine standards were purchased from Sigma‐Aldrich.
Crab meat (3.000 g) was homogenized in 10 ml of 0.4 M perchloric acid (PCA) for 2 min and centrifuged at 5,000 r/min for 15 min at 4℃. After centrifugation, the upper layer was decanted and transferred into a 25 ml brown flask and diluted. A 1 ml extraction was derivatizated by 100 µl of 2 mol/L NaOH, 300 µl of a saturated solution of NaHCO₃, and 2 ml of 10 mg/ml dansyl chloride. All sample solutions were filtered through 0.22 µm filters prior to analysis, and then, 10 µl of the filtrate was injected into the HPLC. Two different eluents were used: (A) 0.01 mol/L ammonium acetate and (B) 90% acetonitrile in 0.01 mol/L ammonium acetate. The wavelength of the detector was set to UV 254 nm.

INS concentration was analyzed using high-performance liquid chromatography (HPLC) as previously reported (Shrestha et al., 2016 ). Fifty microliters of each sample were injected into an HPLC system (e2695; Waters, Milford, MA, USA) consisting of a pump (W2690/5; Waters), an ultraviolet (UV) detector (W2489; Waters), and a data station (Empower 3; Waters). A C18 column was used (ZORBAX 300SB; 150 × 4.6 mm, 5 μm; Agilent Technologies, Santa Clara, CA, USA), and the mobile system consisted of A [0.1% trifluoroacetic acid (TFA) in distilled water] and B (acetonitrile), with a varied gradient according to the following program: 0 min (80% A), 5 min, (50% A), 7 min (50% A), and 10 min (80% A). Analyses were performed at a flow rate of 1.2 mL/min at 35 °C, and the column eluent was monitored at 214 nm.