Extensive evaluation of sample preparation workflow for gas chromatography-mass spectrometry-based plasma metabolomics and its application in rheumatoid arthritis

GC-MS platform has been proved to be an important analytical technique for plasma metabolomics , but lack of efficient sample preparation strategies prior to sample injection has limited its wide application. In the present work, twenty two extraction protocols including protein precipitation (PPT), liquid-liquid extraction (LLE), solid-phase extraction (SPE) and ultrafiltration were simultaneously evaluated using non-spiked and metabolite standards spiked human plasma. Sample-to-extraction solvent ratio and metabolites derivatization conditions were also investigated and optimised. The results demonstrated that stepwise LLE protocol (MeOH–H 2 O/CHCl 3 /CHCl 3 –MeOH) was the most efficient extraction strategy for the combination of untargeted and targeted metabolomics. Sample-to-extraction solvent ratio of 1:3 (v:v) was found to perform better than higher solvent ratios. Also, the maximum derivatization performance was obtained when using 30 μL N-methyl-N-tri-methylsilyltrifluoroacetamide (MSTFA) and 1% trimethyl-chlorosilane (TMCS) incubated at 60 °C for 120 min. In the end, the optimised sample preparation method was applied successfully to plasma untargeted metabolomics of rheumatoid arthritis (RA) and four metabolites (gamma-butyrolactone, MG (0:0/18:0/0:0), tocopherol and oxalic acid) were found to be potential biomarkers for the diagnosis of RA.