Exclusion of Spectral Regions

Usually, one of the first steps of data pre-processing is an exclusion of spectral regions, which contain non-reproducible information or which do not contain information about metabolites. For the data analysis, spectral regions not populated by endogenous metabolites are not useful. As these regions are sensitive to spectral artifacts such as inadequate phasing, exclusion is beneficial. Therefore, the spectrum outside the window of 0.2 ppm to 10 ppm is usually excluded. Other parts of the spectrum, which are usually excluded, are resonances of the solvent water. Although special suppression techniques for water resonances are used, the remaining signal of water, which varies due to different pre-saturations, dominates the spectrum between 4.6 ppm and 5.0 ppm rendering the analyses of signals of metabolites lying below the water resonances impossible. For spectra of urine, the signal of urea, which is very close to water, is the most dominating peak besides of water, as urea is the most concentrated metabolite in urine. Yet, the urea peak is not quantitative, as the protons exchange with water and consequently the peak intensity varies with the quality of water suppression. As this peak has a high intensity and a broad range between 5.4 ppm and 6.0 ppm it highly influences methods of multivariate data analysis unless the peak is excluded.

In many publications about the analysis of urine samples, the exclusion regions of water and of urea are combined and the complete spectral region between 4.50 ppm and 5.98 ppm is excluded. Although this combination of exclusion areas also excludes spectral regions with signals of metabolites of interest (for example the resonance of the α-anomeric proton of glucose at 5.24 ppm), it is the de-facto standard in literature. Consequently, only the spectral ranges between 0.2 ppm and 4.5 ppm and between 5.98 ppm and 10 ppm are analyzed in the case of urine samples. For plasma samples and extracts of tissue the spectral ranges from 0.2 ppm to 4.6 ppm and from 5.0 to 10 ppm are used, whereby additional exclusion regions can be necessary depending on resonances of solvents for tissue extracts.