Assessment of automated trimethylsilyl derivatization protocols for GC-MS-based untargeted metabolomic analysis of urine

Gas chromatography-mass spectrometry (GC-MS) is a commonly used metabolomic platform for the analysis of urine. A key step in the preparation of samples for GC-MS is derivatization, in particular, methoximation and trimethylsilylation. This paper presents an assessment of automated derivatization protocols for GC-MS-based untargeted metabolomic analysis of rat urine. Automated batch and in-time (a sample ready for injection every 70 min) derivatization protocols were tested using N,O-bis(trimethylsilyl)trifluoroacetamide and N-methyl-N(trimethylsilyl)trifluoroacetamide. Principal component analysis determined differences based upon protocol tested (PC-1, 19 %) and silylation reagent (PC-2, 17 %) used. Of 249 compounds, 40 compounds were significantly different (P < 0.05) based upon reagent and 154 compounds were significantly different (P < 0.05) based upon protocol. This study confirms that derivatization reagent and protocol are key factors affecting the reproducibility and intensity of individual urinary metabolites. Additionally, we have identified the majority of the compounds in urine at least to class level, scoring the identifications using the proposed metabolite identification metrics.