Leishmania Fatty Acid Profiling by GC-MS

Parasite fatty acids were characterised and quantified by derivatisation to their fatty acid methyl esters (FAME) followed by gas chromatography-mass spectrometry analysis. Briefly, mid-log phase Leishmania were collected by centrifugation, washed in PBS and freeze-dried in glass tubes. Triplicate aliquots (equivalent to 2×10⁸ cells) were transferred to 2 ml glass vessels, spiked with an internal standard fatty acid C17:0 (20 µl of 1 mM) and dried under nitrogen. Base hydrolysis to release fatty acids was performed using 500 µl of concentrated ammonia and 50% propan-1-ol (1∶1), followed by incubation for 5 hr at 50°C. After cooling, samples were evaporated to dryness with nitrogen and dried twice more from 200 µl of methanol: water (1∶1) to remove all traces of ammonia. The protonated fatty acids were extracted by partitioning between 500 µl of 20 mM HCl and 500 µl of ether. The aqueous phase was re-extracted with fresh ether (500 µl) and the combined ether phases were dried under nitrogen in a glass tube. The fatty acids were converted to FAME, by adding diazomethane (3×20 µl aliquots) to the dried residue, while on ice. After 30 min, samples were allowed to warm to room temp and left to evaporate to dryness in a fume hood. The FAME products were dissolved in 10–20 µl dichloromethane and 1–2 µl analysed by GC-MS on Agilent Technologies (GC-6890N, MS detector-5973) with a ZB-5 column (30 M × 25 mm × 25 mm, Phenomenex), with a temp program of 70°C for 10 min followed by a gradient to 220°C at 5°C/min and held 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 with a bacterial FAME standard that contained both C17Δ and C19Δ (Supelco).