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The application of more than one analytical platform is a prerequisite to success in metabolomics in order to address large dynamic range and chemical diversity of metabolites in body fluids. The combination of LC-ESI-TOF-MS and flow injection NMR can successfully meet these analytical needs. This approach was taken in a medium scale study on human metabolism based on 1000 urine samples where we were interested in identifying influences of lifestyle (in particular smoking) on the metabolic profile as well as on methodological aspects.
Spot urine samples were automatically prepared in 96-well plates for LC-MS (1-fold dilution with water) and NMR (buffered to pH=7. The MetabolicProfiler system consists of a micrOTOF-ESI-TOF mass spectrometer coupled to an HPLC system and a Bruker Avance 600 MHz NMR spectrometer using flow injection technology. The oracle based sample administration and sample order management system SampleTrack controlled the completely automated sample preparation and data acquisition. Data evaluation of LC/MS and NMR data was performed in an integrated approach enabling all steps required for a statistical treatment of spectroscopic data.
The robustness and validity of the combined measurement concept on base of the integrated platform run under full automation could be demonstrated. In total 1134 urine samples were obtained from four different groups: smoker, stop-smoker, nonsmoker and people with unknown smoking status. In a first data screening step based on PCA, several influences typical for metabonomics based population studies were identified, e.g., smoking (nicotine and related metabolites detected by LC/MS), drug intake (drug and related metabolites detected by LC/MS and NMR), diet, age, alcohol abuse and indications on diabetes and latent inborn errors (NMR). More advanced statistical analysis and covariance analysis reveal indication on metabolic changes due to smoking status.