Gas Chromatography-Mass Spectrometry

Lyophilised samples (a total of 66 footprint samples and 66 fingerprint samples) were chemically derivatised by addition of an O-methylhydroxylamine solution (50 μL, 20mg.mL⁻¹ in pyridine 60°C for 30 min) (Sigma-Aldrich, Gillingham, UK); followed by addition of 50μL MSTFA (N-acetyl-N-(trimethylsilyl)-trifluoroacetamide) (Sigma-Aldrich, Gillingham, UK) and heating at 60°C for 30 min. 20μL of a retention index (RI) solution (0.6 mg mL⁻¹ C₁₀, C₁₂, C₁₅, C₁₉ and C₂₂ n-alkanes) was added to the derivatised solution. Particulate matter was removed by centrifugation (15 min, 13,3639g) followed by transfer of the supernatant to 300μl glass inserts placed in 2mL chromatography vials which were sealed with a PTFE/rubber septum containing screw cap.

Derivatised samples were analysed on a 6890 gas chromatograph and 7890 autosampler (Agilent Technologies, Cheadle, UK) coupled to a Pegasus III electron impact mass spectrometer (Leco, Stockport, UK) as previously described³².

Raw data files (.peg format) acquired from the GC–ToF–MS platform were directly processed by applying the ChromaTof software (Leco Corp. v2.25) as previously described³³. Data for each sample set (metabolic fingerprint and metabolic footprint) were integrated as a single dataset in .xls format for further data processing and analysis. Median values were calculated for data acquired for six technical replicates related to a single subject. All data were normalised to the total peak area ([peak area-metabolite/total peak area-all metabolites]*100). Metabolites were identified by comparison of retention index (RI) and electron impact-derived fragmentation mass spectrum to an in-house mass spectral library ³⁴ or by comparison of the mass spectrum to the Golm Metabolome Database (GMD) ³⁵ or NIST08 mass spectral library (http://www.nist.gov/srd/nist1a.cfm). Four different levels of reporting metabolite annotation or identification are available, as defined by The Metabolomics Standards Initiative (http://msi-workgroups.sourceforge.net)³⁶. Level 1 identification was achieved if matching of RI and mass spectrum to a metabolite in the in-house library was achieved. Level 2 identification was achieved by matching to a metabolite present in GMD or NIST08 libraries by mass spectrum only.