Agilent 7890a series gc system

Samples were analyzed and identified using an Agilent 7890A Series GC system coupled with an Agilent 5975C Mass Selective Detector (Agilent Technologies, Santa Clara, CA, USA). Helium was used as the carrier gas at a flow rate of 1.0 mL/min with an HP-INNOWAX capillary column (30 m × 250 µm × 0.25 µm). The inlet temperature was 200 °C. The column temperature program of the GC was initially set at 40 °C for 3 min, heated to 100 °C at 3 °C/min, maintained for 5 min, increased to 200 °C at 20 °C/min, and maintained for 7 min. Mass spectrometry detection used an electron impact (EI) ionization system at 70 eV, the ionization source temperature was 280 °C, the quadrupole temperature was 150 °C, the full-scan acquisition mode was performed with a mass range of 33–350 (m/z), and the tuning file included standard tuning. Constituents were identified by comparing the mass spectra with the National Institute of Standards and Technology (NIST08, http://webbook.nist.gov/chemistry/ (accessed on 29 November 2022)) library and published data. Furthermore, the retention time (RT) of the standard substances for the representative compounds was measured according to the above experimental conditions. Relative percentages (RPs) were calculated to determine the proportion of each component.

Total lipid of seal blubber, whole krill, and penguin muscle, was extracted following a modified Folch et al. (1957 (link)) method (Budge et al. 2006 (link)). Briefly, approximately 0.2–0.5 g of tissue was extracted using 2:1 chloroform: methanol with 0.01% of butylated hidroxytoluene, washed in a salt solution, centrifuged, dried over anhydrous sodium sulphate and evaporated under nitrogen. FA methyl esters were prepared using H₂SO₄ in methanol and then extracted into hexane (50 mg/ml).
Gas chromatography analyses were performed with Agilent 7890A Series GC System (Agilent Technologies, USA) equipped with a flame ionization detector, as described in Guerrero and Rogers (2017 (link)). Identification of FAs and isomers was conducted using known standard mixtures (Nu Check Prep., Elysian, MN, USA). Once FAs were identified, their concentrations were converted to percentage contributions of the total FAs.

Total lipid was extracted as reported [24 (link)]. After 12 days of incubation, the wet biomass is harvested by centrifuging at 5000 rpm and dried at 60 °C. Around 1 g of dry biomass of each experimental set was crushed using mortar and pestle, and the crushed biomass was mixed with 1 mL of chloroform along with 2 mL of methanol, which was then kept for shaking at 150 rpm for 12 h. The mixtures were again shaken at 150 rpm for 1 h after adding 1.5 mL of distilled water, to separate the mixture into bi-phasic layers. From this bi-phasic layer, the bottom layer of chloroform was separated and filtered out using Whattmann filter paper which was then evaporated by Nitrogen flushing. Transesterification of neutral lipid was done using 2 M methanolic-KOH, added in the ratio of 200 µL per 20 mg of total lipid. Heptadecanoic acid methyl ester was added as an internal standard at a concentration of 200 µg/mL to each of the samples for quantification of the fatty acid methyl ester (FAME) content. Transesterified neutral lipids were extracted by 1 mL n-Hexane (HPLC grade) for performing GC–MS. For quantification of FAME, Agilent 7890A series GC system (Agilent Technologies; Singapore) comprising an Omega Wax 250 column (30 m × 0.25 mm 0.25 µm) coupled with an Agilent 7000 QQQ MS was used. All essential parameters for GC–MS experiments are included in Additional file 1: Methods section.