Structure Elucidation - Influences of Study Protocol

The samples of the ranking study were measured by NMR. The first intersting observation was a 10-fold dilution of post-dosed samples. For NMR measurements, a 10 fold dillution needs a 100-fold measurement time for achieving the same signal to noise ratio. All spectra were analyzed by a PCA. Thereby the spectra clusterd into pre-dose and post-dose samples. As the samples of the control group also clustered into the corresponding groups the separation into these two groups cannot be attributed to a common pharmacological effect. The separation could be traced back to a down-regulation of citrate cycle intermediates for the post-dosed samples. Together with the extreme dilution of the post-dose samples, the origin of this separation could be identified as a 24 hour food restriction before sampling the post-dose samples. A 24 hour food deprivation for rats corresponds to several days fasting of humans massively influencing metabolic profiles. Therefore, only post-dose samples were used for further analyses.

PCA of the samples from the ranking study. The samples cluster in the pre-dose and post-dose time point including the control animals each. The reason for this unwanted clustering is a study protocol not suited for metabonomic studies.