Labsolutions software version 5

Muscle samples obtained from the frozen fish (− 80 ºC) were used for the analysis. Those muscle samples of three fish from each tank were combined and homogenized together with 0.1 M HCl in 1:9 (w/v) and spun at 12,000g (4 ºC, 15 min). Supernatants were collected, filtered (Milipore, 10 kDa cutoff at 15,000g, 4 ºC, 30 min) and later diluted with 0.1 M HCl (1:19 v/v) containing norvaline and sarcosine (40 µM) as internal standards. Blanks (0.1 M HCl + 40 μM norvaline and sarcosine) and external standards (Sigma acid/neutral and basic amino acids) were prepared along with the sample preparation. The same concentration of glutamine in 0.1 M HCl as an external standard was prepared and added to the basic amino acids standard. Free amino acids were quantified using Shimadzu Prominence Nexera—i LC-2040C Plus (Shimadzu, Japan) according to the Shimadzu protocol No. L529 with modifications³⁰ . Free amino acid concentrations (expressed as µmol/kg wet body weight) were calculated in LabSolutions software version 5.92 (Shimadzu, Japan, https://shimadzu.com.au/labsolutions) using internal and external standards.

FAA analysis of fish tissues was performed according to Kwasek et al. [27 (link)]. Muscle samples of three fish from each tank were combined and homogenized together with 0.1 mol/L HCl in 1:9 (w/v) and spun at 12,000× g (4 °C, 15 min). Supernatants were collected, filtered (Milipore, 10 kDa cutoff at 15,000× g, 4 °C, 30 min), and later diluted with 0.1 mol/L HCl (1:19 v/v) containing norvaline and sarcosine (40 μmol/L) as internal standards. Blanks (0.1 mol/L HCl + 40 μmol/L norvaline and sarcosine) and external standards (Sigma acid/neutral and basic AA) were prepared along with the sample preparation. The same concentration of glutamine in 0.1 mol/L HCl as an external standard was prepared and added to the basic AA standard. Free amino acids were quantified using Shimadzu Prominence Nexera—i LC-2040C Plus (Shimadzu, Japan) according to the Shimadzu protocol No. L529 with modifications. Free amino acid concentrations (expressed as μmol/kg wet body weight) were calculated in LabSolutions software version 5.92 (Shimadzu, Japan) using internal and external standards.

Chromatographic analyses were performed on HPLC-DAD-UV using a Shimadzu Nexera XR^® liquid chromatographer coupled to a Shimadzu UV detector with diode array SPDM20A, equipped with a CBM20A controller, DGU20A degasser, LC20AD binary pump, CTO20A oven, and SILA20A auto-injector. A Shimadzu LabSolutions Software Version 5.3 (Shimadzu, Kyoto, Japan) was used to analyze chromatograms. Combinations of acidified ultrapure water (pH 3.0, with anhydrous acetic acid, Merck, Darmstadt, Germany) (A) and acetonitrile (HPLC grade, Tedia, Rio de Janeiro, Brazil) (B) were used as the mobile phase (initially 5% A rising to 95% in 80 min). HPLC column was silica-based C18 (250 mm × 4.6 mm i.d. × 5 μm particle size, ODS Hypersil, Thermo, Waltham, MA, United States). The oven was set at 50°C and the injection volume was 10 μL for all analyses.

Chromatographic analyses were performed on a high-performance liquid chromatograph coupled with a diode-array UV-Vis detector (HPLC-DAD-UV), using a Shimadzu Nexera XR^® liquid chromatograph coupled to a Shimadzu, Kyoto, Japan, UV detector with the diode array SPDM20A, equipped with a CBM20A controller, DGU20A degasser, LC20AD binary pump, CTO20A oven and SILA20A auto-injector. A Shimadzu LabSolutions Software Version 5.3 (Shimadzu, Kyoto, Japan) was used to analyze chromatograms. DAD analysis was applied to select the optimized wavelength of anthocyanidins in this study. In a full-scan experiment, chromatograms at 480 nm show the maximum wavelength (λ_max) for the anthocyanidins. Combinations of acidified ultrapure water (pH 3.0, with anhydrous acetic acid, Merck, Darmstadt, Germany) (A) and acetonitrile (HPLC grade, Tedia, Rio de Janeiro, Brazil) (B) were used as the mobile phase (initially 5% A rising to 95% in 80 min). HPLC column was silica-based C18 (250 mm × 4.6 mm i.d. × 5 μm particle size, ODS Hypersil, Thermo, Waltham, MA, USA). The oven was set at 50 °C and the injection volume was 10 μL for all analyses.

Chromatographic analyses were performed on a HPLC-DAD-UV using a Shimadzu Nexera XR^® liquid chromatograph (Shimadzu, Kyoto, Japan) coupled to a UV detector with an SPDM20A diode array, a CBM20A controller, a DGU20A degasser, an LC20AD binary pump, a CTO20A oven, and an SILA20A auto-injector. Shimadzu LabSolutions Software Version 5.3 (Shimadzu, Kyoto, Japan) was used to analyze the chromatograms. DAD analysis was applied to select the optimized wavelength of the meroterpenoids in this study (300 nm). Combinations of ultrapure water (A) and methanol (HPLC grade, Tedia, Rio de Janeiro, Brazil) (B) were used as the mobile phase (initially 0% B, increasing to 20% in 8.5 min, subsequently rising to 100% of B in 68.5 min, and finally staying at this concentration up to 90 min). The HPLC column was silica-based C18 (250 mm × 4.6 mm i.d. × 5 μm particle size, Shimpack CLC-ODS, Thermo, Waltham, MA, USA). The oven was set to 50 °C, and the injection volume was 10 μL for all analyses.

A solution was prepared by dissolving 100 mg of crude plant extract in 10 mL of methanol. The resulting solution was then diluted to a concentration of 2 mg/mL using a mixture of 50% methanol and high-purity water. The solution was subsequently passed through a 0.22 mm scale filter before being transferred to a bottle for LC-ESI/MS/MS (Liquid Chromatography and Mass Spectrometry) analysis. The Poroshell 120 EC-C18 (Agilent Technologies, Santa Clara, CA, USA) column was used to chromatographically isolate various components. For mass spectrometric detection, the Shimadzu LCMS-8040 model mass spectrometer was used. The LC-ESI-MS/MS data were analyzed using the LabSolutions software (Version 5.3) developed by Shimadzu (Kyoto, Japan). Phytochemicals were measured using Multiple Reaction Monitoring (MRM) technique. Research using mass spectrometry (MS) was carried out with specific experimental parameters [66 (link)].

Dodecyl stearate (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) served as the standard during analysis of wax esters. An HPLC platform (30A (Nexera X2) series), an MS/MS unit (LCMS-8060 series), and Labsolutions software version 5.86 from Shimadzu Corp. were used for measurements. A calibration curve was prepared from a linear regression equation (using the least-squares method) of the peak area ratios of the calibration curve samples. The sample values were calculated by fitting the peak area ratios of the sample solutions and recovered samples to the calibration curve. If wax esters other than dodecyl stearate were detected, the measured values were calculated using the calibration curve of dodecyl stearate.