7683b series injector

Percentages of total fatty acids from placental lipids (including glycerolipids, phospholipids, sphingolipids, sterol lipids, and free fatty acids) were analyzed as fatty acid methyl ester derivatives (FAMEs) by gas chromatography (GC) as previously described [57 (link)]. Briefly, lipids from 500 mg of placental samples were extracted with chloroform/methanol (2:1, v/v) in the presence of 0.01% butylated hydroxytoluene. The fatty acids were trans-esterified and the resulting fatty acid methyl esters were extracted. A total amount of 4 μL were used for GC analysis. Separation was performed with a DBWAX capillary column (30 m × 0.25 mm × 0.20 μm) in a GC System 7890A with a Series Injector 7683B and an FID detector (Agilent Technologies, Barcelona, Spain). Identification of fatty acid methyl esters was made by comparison with authentic standards (Larodan Fine Chemicals, Malmö, Sweden). Results are expressed as mol percentage (mol %) [56 (link)].
The following fatty acids groups were determined: saturated fatty acids (SFA); monounsaturated fatty acids (MUFA); polyunsaturated fatty acids (PUFA) from n-6 and n-3 series (PUFAn-6 and PUFAn-3, respectively); and the following ratios were also calculated: LA/ALA, AA/EPA, AA/DHA, AA/EPA + DHA, and the n-6/n-3 PUFA ratio as the quotient between all the PUFA n-6s divided by all the PUFA n-3s.

Briefly, samples were incubated for lipid extraction and FAs transesterification in 2 ml of 5% methanolic HCL at 75 °C for 90 min. FAs methyl esters were extracted by adding 2 ml of n‐pentane and 1 ml of saturated NaCl solution. Samples were separated and evaporated under N2 gas n‐pentane phase and finally dissolved in 80 µl of carbon disulphide. Gas chromatography (GC) analysis was then performed.
The GC method was used for separation with a DBWAX capillary column (30 m × 0.25 mm ×0.20 μm) in a GC System 7890 A with a Series Injector 7683B and an FID detector (Agilent Technologies, Barcelona, Spain). The temperature of the injector was 220 °C using the splitless mode. A constant rate (1.8 ml/min) of helium (99.99%) was maintained. The column temperature was held at 145°C for 5 min; subsequently, the column temperature was increased by 2°C/min to 245°C for 50 min, and held at 245°C for 10 min, with a post‐run of 250°C for 10 min as previously described [59, 60, 61]. Based on FA composition, different indexes were calculated, and elongase and desaturase activity was estimated from specific product/substrate ratios [61, 62].

Fatty acids from mitochondrial membranes were analyzed as methyl ester derivatives by gas chromatography (GC) as previously described [25 (link)]. Separation was performed by a DBWAX capillary column (30 m × 0.25 mm × 0.20 μm) in a GC System 7890A with a Series Injector 7683B and an FID detector (Agilent Technologies, Barcelona, Spain). Fatty acid methyl esters were identified by comparison with authentic standards (Larodan Fine Chemicals, Malmö, Sweden). Results are expressed as mol%. The following fatty acyl indices were also calculated: saturated fatty acids (SFA); unsaturated fatty acids (UFA); monounsaturated fatty acids (MUFA); polyunsaturated fatty acids (PUFA) from n-3 and n-6 series (PUFAn-3 and PUFAn-6); and average chain length (ACL) = [(Σ%Total14 × 14) + (Σ% Total16 × 16) + (Σ% Total18 × 18) + (Σ% Total 20 × 20) + (Σ% Total 22 × 22) + (Σ% Total 24 × 24)]/100. Finally, the density of double bonds in the membrane was calculated by the Double Bond Index, DBI = [(1 × Σmol% monoenoic) + (2 × Σmol% dienoic) + (3 × Σmol% trienoic) + (4 × Σmol% tetraenoic) + (5 × Σmol% pentaenoic) + (6 × Σmol% hexaenoic)].