5. Tissue metabolomics
Preparation of tissue samples is a very time-consuming, labor intensive procedure. In particular for global metabolomics, when the primary goal is extraction of the highest possible number of metabolites, a multi-step solvent extraction is required to recover both hydrophobic and hydrophilic species. Comparative studies of six solvent-based sample-preparation protocols for LC-MS analysis showed that the most efficient method in terms of reproducibility and number of metabolite features obtained used methanol/ water for extraction of polar compounds with subsequent use of a dichloromethane/methanol mixture for recovery of non-polar compounds [7]. The two-step extraction was followed by re-suspension of the dried extracts in methanol/water. The protocol offers com- prehensive analyte coverage of a few thousand molecular features. However, this coverage involves a compromise with the total time of the experiment. The overall time of the final sample prepara- tion approach proposed by the same group was ~8–10 h using a 96- well plate format [81]. For the analysis of organs, where collection of biopsy is relatively easy (e.g., liver), the standard protocols can be effectively used, while, in the situations of tissue shortage (e.g., problem with sample collection, need for repeated analysis, and danger of side effects related to tissue collection), there is a need for other analytical approaches able to overcome the challenge of the time required. As explained before, SPME can be used as an ex- traction method for ex vivo/in vitro analysis, and, when applied to in vivo analysis, it allows for the omission of a sample-collection step. Due to the small dimension of the probe, SPME offers minimal invasiveness and no side effects. To date, in vivo tissue metabolomics by DI-SPME analysis reported in the literature were performed using the mix-mode coating (C18 and benzenesulfonic acid) [6,74]. In both cases, coverage of analytes was not comparable to the one reported for solvent-based methods, but the investigations exhibited benefits of the technology not available with standard methods.