Metabolite extraction and derivatization

A total of 5 triplicated MCF-7 cell culture samples were provided in Eppendorf vials dissolved in PBS, and stored at 4 °C for preservation purposes. Samples were centrifuged at 13000 rpm for 10 minutes at −4 °C. Supernatant was discarded, and cell pellets, each containing 3 million cells, were subjected to metabolomics analysis.

In order to evaluate the influence of the solvents on the extraction rate, samples were divided in five different extraction groups: A, 0.2% formic acid in water; B, 0.2% ammonium hydroxide in water; C, ethyl acetate; D, methanol/water (1:1, v/v); and E, acetonitrile/water (1:1, v/v).

Metabolic content extraction of the adenocarcinoma cells (MCF-7), applied in this research, was based on optimized method by our research group. Briefly, 300 µL of the extraction solvent was added to 3 million cell pellets, then vortexed for 2 minutes. To ensure the quantitative extraction of the metabolites, samples have been stored in ice for 1 hour, during which samples have been vortexed every 15 minutes. After this, cell insoluble matrix was centrifuged (13000 rpm, 10 minutes, −4 °C). Supernatant was collected and transferred to GC vial, then dried using EZ-2 Plus (GeneVac, Ipswich, UK) at 37 ± 1 °C. Amino acids, saccharides, fatty acids and steroids cannot be analysed directly by gas chromatography due to their high polarity and low volatility. Hence, it was necessary to derivatize them prior to the GC-MS analysis. Having said that, dry samples were dissolved in 25 µL of a solution consisting 20 mg/mL methoxyamine hydrochloride in pyridine, followed by vortexing for 2 minutes, and storing for at least 6 hours at 25 °C prior to the silylation step. Then 25 µL of MSTFA + 1% TMCS was added, followed by dissolving in 100 µL of pyridine, and vortexing for 2 minutes. For complete derivatization, it was necessary to incubate the samples at 50 °C for 30 minutes. Due to low volume, all of the samples have been immediately transferred to 200 µL microinserts, and analysed by GC-MS.