Empower software

Metalaxyl analysis was performed by WATERS 2695 Separations Module HPLC system with Empower^TM software equipped with a sample manager, a quaternary solvent manager connected to UV detector (Waters Co., MA, USA) and SunFire^TM C18, and a 5 µm column (4.6 mm × 150 mm) (Waters Co., MA, USA). Ten microliters of the sample were injected using the auto-injector with a 100 µL injection loop for the total analytical time of 12 min. The mobile phase was ACN: 0.1% formic acid in water and a flow rate of 1 mL/min with gradient elution. The following mobile phase gradient was used: 0–2 min, 55% ACN, 2–5 min, ACN was increased from 55% to 65%, 5–7 min held at 65% ACN, 7–7.5 min, ACN was decreased from 65% to 55%, and 7.5–12 min, held at 55% ACN. The detection wavelength was set at 220 nm.

Hydrolyzed microalgae samples (6 M HCl solution at 116 °C for 48 h) were precolumn derivatized with Waters AccQ Fluor Reagent (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate) using the AccQ Tag method (Waters, Milford, MA, USA). Analyses were performed by ultra-high-performance liquid chromatography on a Waters reversed-phase amino acid analysis system. Norvaline was used as internal standard. The resulting peaks were analyzed with the EMPOWER software (Waters) [16 (link)]. Analyses were performed in duplicate. Amino acid scores (AAS) were calculated [17 (link)], using the minimum levels recommended by FEDIAF [18 ] for complete food for adult dogs (maintenance energy requirement of 110 kcal/kg^0.75), dogs in early growth (<14 weeks) and reproduction, and late growth (≥14 weeks). The geometric mean of AAS was calculated to determine the index of essential amino acids [19 ].

The lyophilized extract (10 mg) was dissolved in 70% methanol (5 mL) and then filtered through a 0.2 μm membrane filter (Woongki Science Co., Ltd., Seoul, Korea) before being injected into HPLC for component analysis. The ferulic acid (Sigma-Aldrich; purity over 98.0%) was used as a standard indicator of CoM in this study [17 (link)]. The HPLC-grade solvents, methanol, acetonitrile, and water were obtained from J. T. Baker (Phillipsburg, NJ). Trifluoroacetic acid (analytical reagent grade) and the standards were procured from Sigma-Aldrich. The HPLC system consisted of a Waters Alliance 2695 system coupled with a 2998 photodiode array detector (Waters Co., Milford, MA). Data processing was performed with Empower software (version 3; Waters Co.). The ferulic acid in CoM was separated using a Luna 5 μm C₁₈ 100 A column (4.6 × 250 mm, 5 μm particle size; Phenomenex Inc., Torrance, CA). The monitoring was performed at 240 nm for ferulic acid. The mobile phases consisted of water with 0.1% (v/v) distilled water (solvent A) and Trifluoroacetic acid (solvent B) at a flow rate of 1.0 mL/min. The gradient conditions changed as presented in Table 1. The injection volume was 10 μL.

Carotenoids were determined according to the method in our previous study (22 (link)). A 0.5 g freeze-dried tomato fruit sample powder was taken and 30 ml of a mixture of acetone and petroleum ether was added (1: 2, v/v). After ultrasonic extraction at 30^°C for 40 min, the extract was dried at 40^°C, then dissolved in a mixture of 6.25 ml methanol, 5 ml dichloromethane, and 13.75 ml acetonitrile. Subsequently, a 2-ml mixture was added through a 0.22-μm organic filter membrane, and the filtrate was used for an Alliance Waters e2695 HPLC (Waters, United States). The chromatographic column: C₁₈ column (250 mm × 4.6 mm, 5 μm, Waters, United States), mobile phase was composed of methanol: dichloromethane: acetonitrile (25:20:50, v/v/v), flow rate: 1.2 ml min^–1, injection volume: 10 μl, and column temperature: 30^°C.
Phytoene (Sigma, United States) was detected at 286 nm, lutein and β-carotene (Yuanye, China) were detected at 450 nm, and lycopene (Yuanye, China) was detected at 470 nm. The retention time of lutein, phytoene, lycopene, and β-carotene was 2.7, 7.7, 8.3, and 12.7 min, respectively. Data were analyzed with Empower Software (Waters, United States).

Intraplatelet serotonin (5-hydroxytryptamine) was measured using a high-performance liquid chromatography (HPLC) technique [15 (link)]. The HPLC system consisted of Ultrasphere 5-μm ODS column, 250 × 4.6 mm (HiChrom, Theale, UK), a Waters 515 HPLC pump (Waters, Milford, MA, USA), a Rheodyne manual injector (Sigma-Aldrich, St. Louis, MO, USA), an electrochemical detector (Waters 464), and EMPOWER software (Waters). A platelet sample (20 μl) was injected for HPLC analysis, and the amount of serotonin was calculated on the basis of a calibration curve.