Collection of optimizations for untargeted metabolomics analysis of Leishmania promastigotes using gas chromatography-mass spectrometry.

There is no consensus in the literature regarding the ideal protocol for obtaining and preparing cell samples for untargeted metabolomics. Nevertheless, the procedures must be carefully evaluated for proper and reliable results for each organism under study. This work proposes a novel protocol for determining intracellular metabolites in Leishmania promastigotes and is fully optimized for application in conjunction with gas chromatography-mass spectrometry platforms. Sample harvesting consisted of stopping metabolic activity by placing the parasite cells in a dry ice bath and removing extracellular interferants with two wash steps using cold PBS. The extraction is carried out with 1.0x108 promastigotes per sample using a mixture of cold 1:1 methanol:water and ultrasound mixing (1 min at 30 % power). Dried extracts were derivatized by oximation (at room temperature for 90 min), followed by silylation (at 40 °C for 30 min). The method developed here can cover a wide range of the Leishmania parasite metabolome, including amino acids and derivatives, organic and fatty acids, carbohydrates and derivatives, and steroids.