Why my metabolite concentrations deviate from predicted concentrations?

Metabolite intensities (peak areas) depend on several factors, such as ion suppression effects, caused by the sample’s matrix, metabolite dilution or extraction effects, e.g. the metabolite extraction efficiency from a cell pellet, or the ratio of solvent to the cell number, or the ionization efficiency, which is influenced by the sum of all chemical compounds of a sample.
Therefore, metabolite intensities (peak areas) are normalized to internal standard intensities (peak areas).
The quotient of the metabolite and internal standard intensities (area ratio) is less influenced by matrix and ionization effects.
Dilution factors, e.g. the ratio of solvent to the cell number, is not normalized by using internal standards, but should be normalized using one of these two WebIDQ tools.
→ Tissue and cell factors.

Philosophical concluding remark
The result of an experiment may be unpredictable. If the results of an experiment do not comply with the assumptions, research may be the key to identify the source of discrepancies between the assumptions and the experimental results.