Boron trifluoride methanol solution 14

Glyceryl triheptadecanoate (2 μg) as internal standard and hexane (1 mL) was added to the lipid extract (80 μL from the total 400 μL). Transmethylation was performed for 1 hour in boron trifluoride methanol solution 14% (1 mL, Sigma-Aldrich) at 55°C. After the addition of water (1 mL) to the crude, fatty acid methyl esters (FAME) were extracted with hexane (3 mL), dried by evaporation, and dissolved in ethyl acetate (20 μL). FAME (1 μL) were analysed by gas-liquid chromatography [32 (link)] on a 5890 Hewlett Packard system using a Famewax RESTEK-fused silica capillary columns (30 m × 0.32 mm i.d., 0.25 mm film thickness). Oven temperature was programmed from 110°C to 220°C at a rate of 2°C per min, and the carrier gas was hydrogen (0.5 bar). The injector and the flame ionization detector were at 225°C and 245°C, respectively.

Acetonitrile (ACN) was HPLC-grade and purchased from Acros Organics (Geel, Belgium). Methanol HPLC-grade (MeOH), Dichloromethane (CH₂Cl₂), Ammonium Formate (>99%) (AF), Boron trifluoride-methanol solution 14% (BF3-MeOH), Heptane, Ethyl acetate (EtOAc), potassium hydroxide (KOH), pentafluorobenzyl bromide (PFB-Br), and diisopropylethylamine (DIPEA), iodoacetamide, ammonium bicarbonate, trifluoroacetic acid, and trypsin was supplied by Sigma Aldrich Chemicals Co. (Saint Quentin Fallavier, France), acetic acid (AA) from Honeywell Fluka. Ultrapure water (18.2 MΩ) was obtained from a milliQ apparatus from Millipore (Guyancourt, France).
Internal synthetic standards of phospholipids (PL: PE 12:0/12:0, PC 13:0/13:0, PS 12:0/12:0), Ceramides (Cer: Cer d18:1/15:0), sphingomyelins (SM: SM d18:1/12:0), and sphingosine (So: So d17:1) and sphinganine (Sa: Sa d17:0) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Synthetic internal standard PI 16:0/17:0 was supplied by J. Clark (Cambridge). Synthetic internal standards for neutral lipid (LN: stigmasterol, cholesteryl heptadecanoate, glyceryl trinonadecanoate) and for free FAs (FFA: heptadecanoate) and total FAs (TFA: glyceryl triheptadecanoate, glyceryl trinonadecanoate) were purchased from Sigma Aldrich (St Quentin Fallavier, France).