Gc 6890n gas chromatograph

Acid hydrolysis of free fatty acids was performed on the dry total lipid extracts. The reaction (total volume 1mL) is conducted in a glass vial. 100 μL of toluene were added, followed by 750 μL of MeOH and 150 150 μLof 8% HCl MeOH:H₂O 85:15 (v/v) solution in order to allow the esterification of the free fatty acids. The reaction is left to go to completion at 45°C overnight. Upon drying, the fatty acid methyl esters (FAMEs) were extracted with a 1:1 hexane:H₂O. The FAME extracts were dried under nitrogen gas stream. The FAME extracts were dissolved in dichloromethane, typically 20 μL and 1 μL is analysed by GC-MS on an Agilent Technologies GC-6890N gas chromatograph coupled to an MS detector‐5973. Separation by GC was performed using a PhenomenexZB-5 column (30 M x 25 mm x 25 mm), with a temperature program of 70°C for 10 min, followed by a gradient to 220°C, at 5°C/min and maintained at 220°C for a further 15 min. Mass spectra were acquired from 50-500 amu. The identity of FAMEs was carried out by comparison of the retention time and fragmentation pattern against bacterial and mammalian FAME standards and online available FAME library (http://www.lipidhome.co.uk/ms/methesters/me-arch/index.htm).

Transmethylation of FFAs was performed on the dry total lipid extracts. The reaction (total volume 1 ml) is conducted in a glass vial. A total of 100 μl of toluene was added, followed by 750 μl of MeOH and 150 μl of 8% HCl MeOH:H₂O 85:15 (v/v) solution to allow the esterification of the FFAs. The reaction was left to go to completion at 45 °C overnight. Upon drying, the fatty acid methyl esters (FAMEs) were extracted with a 1:1 n-hexane:H₂O. The FAME extracts were dried under nitrogen gas stream. The FAME extracts were dissolved in dichloromethane, typically 20 μl, and 1 μl was analysed by GC–MS on an Agilent Technologies GC-6890N gas chromatograph coupled to an MS detector‐5973. Separation by GC was performed using a PhenomenexZB-5 column (30 M × 25 mm × 25 mm), with a temperature programme of 70 °C for 10 min, followed by a gradient to 220 °C, at 5 °C min⁻¹ and maintained at 220 °C for a further 15 min. Mass spectra were acquired from 50 to 500 a.m.u. The identity of FAMEs was carried out by comparison of the retention time and fragmentation pattern against bacterial and mammalian FAME standards and online available FAME library⁴⁸ .