SMC develops quantitative methods for metabolite analysis to be able to offer new targeted methods for future projects. We accept requests from researchers for specific metabolites with the aim to establish targeted methods. After discussing the client's project, we do a pilot study to determine if it is possible to analyze metabolites in the defined biological tissue (matrix) using either LC-MS or GC-MS. The purpose of the pilot study is to determine if it is possible to continue the method development or not. Then the method development is performed according to a number of analytical requirements. When the project is finished the outcome is compiled as an application note describing the result and is communicated to the customer.
- The method must have a simple (preferably one step) and defined extraction procedure which can be used to generate an extract from the matrix suitable for metabolite analysis by mass spectrometry.
- The metabolites of interest must be stable in the extract until analysis by GC-MS or LC-MS.
- The metabolites must have a unique defined retention time (RT) on a chromatographic column, mass signal (molecular ion or eq.) in a full scan analysis and/or unique transition from a tandem mass analysis (e.g. MRM).
- Internal standards with similar chemical structure and chemical behavior must be available for quantification of the target metabolites.
- There should be a linear response at least in a magnitude order of 103 between the instrumental signal (MS detector) and each concentration of the target metabolites.
- The response of a metabolite must increase when the matrix extract is spiked with its corresponding standard.
- It must be possible to detect the metabolites within the linear concentration range
THE APPLICATION NOTE - documentation of the pilot method development
The outcome from the pilot method development is compiled as an application note used for future target analysis, and can be used as the basis in a publication. The documentation will include:
- A list of relevant references from the scientific literature
- Definition of the utilized matrix (e.g. cell lines or plasma)
- A protocol for sample preparation and extraction of the metabolites from the defined matrix
- A protocol describing instrumental parameters and the analytical procedure
- A chromatogram of standards and internal standards, both in organic solvent and in spiked matrix extract (high and low concentrations)
- Estimated detection limits of target metabolite standards
- Data showing the linear response of the target metabolites
- Data confirming that the metabolites are stable in the extract until the time of analysis
- Results showing the signal of the target metabolites in the defined matrix when they are spiked by a defined concentration of metabolite standards