Agilent 7890a system

Oil samples were analyzed on an Agilent 7890A system (Agilent Technologies, Inc., Santa Clara, CA, USA) equipped with a flame ionization detector (FID) using a 30 m PEG column (Size: 30 m, Ø = 0.25 mm, film thickness 0.25 µm) with hydrogen as the carrier gas at Callisons (Lacey, WA, USA). The flow rate was set at 1 mL min⁻¹ and the oven ramp protocol was set to 60–230 °C over 17 min. Compounds were identified via a retention time library established from in-house gas chromatography/mass spectrometry data. Values represent the area percentage of the entire chromatogram (an example of GC-FID chromatograms is included in Fig. S6).

The percentages of FAs in intramuscular fat were determined by gas chromatography (GC) (10 (link)) in an Agilent 7890A system (Agilent Technologies, Santa Clara, CA, USA). Briefly, lipids were extracted from the PMM tissue using chloroform, and methyl esters were obtained via saponification with a solution containing 2 ml of hexane, 40 μl of methyl acetate, and 100 μl of sodium methoxide. After vortexing, the hexane layer was removed from the solution via anhydrous sodium sulfate, and FAs were determined by GC (chromatographic column sp-2560; 100 m × 250 × 0.2 μm) using the following conditions: initial column temperature, 140°C for 15 min; 3°C/min to 240°C; and 15 min at 240°C. The temperature of the injector and detector was at 250°C and the hydrogen flow rate at 30 ml/min, air at 400 ml/min, nitrogen at 40 ml/min, and carrier gas at 0.8 ml/min. The inlet temperature was 220°C, split ratio was 10:1, and injection volume was 1 μl. Individual FA peaks were identified by comparing their retention times with those of corresponding standards (Sigma Chemicals, St. Louis, MO, USA). Data are expressed as g/100 g of total identified FAs.

Previously we measured the PUFA contents of 269 one-year-old common carp from three strains, including 124 individuals of the Furui strain (FRC), 98 individuals of the Jian strain (JC), and 47 individuals of the Huanghe strain (HHC) [24 (link)]. Briefly, the freeze-dried muscle powders were dissolved in NaOH-methanal and then the boron trifluoride-methanol solution. The fatty acid methyl esters (FAMEs) were dried with the oxygen-free nitrogen and then resuspended with hexane. The solution was analyzed by gas chromatography on an Agilent 7890A system (Agilent Technologies, Santa Clara, CA, USA).

Larval oil was obtained by extraction with a 2:1 chloroform-methanol mixture. Fatty acid methyl esters (FAMEs) of samples were analyzed by gas chromatography (GC) on an Agilent 7890A system (Agilent Technologies, Santa Clara, CA, USA) with a flame ionization detector (GC-FID), auto-injection module for liquid, equipped with a fused silica capillary column (Supelco SP-2560 Capillary GC Column 100 m × 0.25 mm, d = 0.20 μm) (Supelco, Bellefonte, PA, USA) and helium as a carrier gas (purity = 99.9997 vol %, flow rate = 1.5 mL/min and pressure = 1.092 bar). The principle behind the FAMEs is the comparison between retention time and reference standards (Supelco 37 component FAME mix, Sigma Aldrich, Darmstadt, Germany). The final results were expressed as % of individual FA or FA group in total identified FA.

Fatty acids were analyzed following a previously described method^{30 (link)} with some modifications. First, 100 mg of ground frozen pepper seed was placed in a Teflon-cap tube and extracted with 2 mL of methylation mixture (methanol:benzene:2,2′-dimethoxypropane:H₂SO₄ = 39:20:5:2, v:v:v:v) and 1 mL of heptane at 80 °C for 2 h. After cooling to 22 °C, the supernatant was collected and analyzed through GC using the Agilent 7890A system (Agilent). The column was DB-23 (0.25 × 60 × 0.25 μm, Agilent). The flame ionization detector (FID) was set at 280 °C, and the flow rates were 35 mL·min⁻¹ for H₂, 350 mL·min⁻¹ for air, and 35 mL·min⁻¹ for He. The injector temperature was 250 °C. The oven temperature was increased from 50 °C to 130 °C at 15 °C·min⁻¹, 170 °C by 8 °C·min⁻¹, and 215 °C by 2 °C·min⁻¹.

Synechocystis cells from 150 mL of culture were harvested by centrifugation, re-suspended in 10 mL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and then lysed by sonication. The lysate was extracted for 30 minutes at room temperature with 10 mL chloroform-methanol (v/v, 2:1). Prior to extraction, 30 μg 1-pentadecanol, 50 μg eicosane, and 100 μg heptadecanoic acid (Sigma Aldrich, USA) were added into the cell lysate as internal standards (Figure 4). The organic phase was separated by centrifugation and evaporated to dryness under nitrogen at 55°C. The dryness was dissolved in 1 mL of n-hexane and 1-μL aliquot was analyzed by GC-MS using an Agilent 7890A system equipped with a HP-INNOWax (30 m × 250 μm × 0.25 μm, Agilent, USA). Helium (constant flow 1 mL min⁻¹) was used as carrier gas. The temperature of the injector was 250°C. The following temperature program was applied: 100°C for 1 minute, increase of 5°C min⁻¹ to 150°C and then 10°C min⁻¹ to 250°C, hold at 250°C for 15 minutes. The internal standards were used to determine the product yield, which was the mean of three independent experiments [15 (link)].