Famq 005

Lipid content and the lipid profile were determined with one-step in-situ transesterification [43 (link)] and subsequent analysis of the derived Fatty acid methyl esters (FAMEs) was made on a GC (7890A, Agilent Technologies Inc., Santa Clara, CA, USA), equipped with a flame ionization detector (FID) and a capillary column (DB–WAX, 10 m × 0.1 mm × 0.1 μm) as previously described [44 (link)]. To quantify the produced FAMEs, a reference standard (FAMQ-005, Accustandard, New Haven, CT, USA) and an internal standard solution (C17: 0, Sigma-Aldrich, St. Louis, MO, USA) were used [44 (link)].

FAME samples were analyzed by injecting 1 µL of the FAME sample into the GC-FID. Hydrogen was used as the carrier gas at a flow rate of 1 mL/min and a split ratio of 30:1. FAME separation was achieved on a BPX-70 (0.32 mm; film thickness 0.25 µm, length 50 m) silica column (Phenomenex, Torrance, CA, USA). The initial column temperature was 100 °C, which was increased to 160 °C at a rate of 10 °C/min, and then up to 220 °C at a rate of 3 °C/min. This temperature was maintained for 5 min, and then increased to 260 °C at a rate of 10 ºC/min, and then held for a further 5 min. The retention times of 37 fatty acid methyl esters were determined using commercial FAME standards (FAMQ-005, AccuStandard, Inc., New Haven, CT, USA), which were analysed by GC-FID under identical running conditions. Docosapentaenoic acid (DPA) and several unknown peaks were identified based on the reported literature [5 (link),6 (link),7 (link),8 (link)].

We injected 1 µl of each FAME solution in an Agilent Technologies 6890N GC Network System with a 7683 series injector and coupled to an electronic impact-mass spectrometry detector (EI-MS; 5973N MSD; Agilent Technologies). The capillary column used was a BPX70 column (30 m × 0.25 mm i.d., 0.25 µm film thickness; SGE Analytical Science). The injector in split mode (20:1) was set at a temperature of 270°C and the oven was maintained for 5 min at 100°C after injection and then increased to 240°C at a ramp rate of 2.5°C/min. The carrier gas was helium at a flow rate of 1.2 ml/min. The source inlet of the mass spectrometer was held at 230°C and 70 V. The identification and quantification of FAMEs was achieved by comparison to retention times of FAME reference standards (FAMQ-005, AccuStandard, New Haven, USA) and other FAMEs identified before in liver, testis and sperm samples with the same analytical method (Reglero et al., 2009 (link); Castellanos et al., 2010 (link)), and by their mass spectra. Calibration curves were performed with FAMEs concentrations ranging from 0.06 ng/µl to 11.89 ng/µl. Blanks were processed with each batch of samples.