SMC performs development of quantitative methods for metabolite analysis in order to offer new targeted methods for future projects. We accept methodological requests from researchers for specific metabolites with the goal of establishing targeted methods. Following a discussion of the client's project, we carry out a pilot study in order to determine if it is possible to analyze metabolites in the defined biological tissue (matrix) using either LC-MS or GC-MS. For these pilot studies we will to as great extent as possible use our existing workflow (methanol / water or Folch extraction, UHPLC (C₁₈)-ESI-QqQ- or QTOF-detection, alternatively GC-TOF or GC-QqQ detection). The goal of the pilot study is not to produce a fully validated analytical method but to discern whether it is feasible to continue the method development or not. The method development at SMC is performed according to a number of analytical requirements. After the project is finished, the outcome is compiled as an application note describing the result and is communicated to the customer.

See this example. (Cancer Res July 2016 DOI: 10.1158/0008-5472.CAN-15-291)

REQUIREMENTS FOR A TARGETED METHOD FOR ANALYSIS OF METABOLITES IN A DEFINED SAMPLE MATRIX

• 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).
• For quantification of the target metabolites, internal standards with similar chemical structure and chemical behavior must be available.
• 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

DOCUMENTATION OF THE PILOT METHOD DEVELOPMENT (the application note)
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 (either MeOH or Folch (or eq.)  extraction will be tested)
• A protocol describing instrumental parameters and the analytical procedure
• A chromatogram for the standards and the internal standard, both in organic solvent and in spiked matrix extract (high and low concentrations)
• Estimated detection limits for target metabolites 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 for the target metabolites in the defined matrix when they are spiked by a defined concentration of metabolite standards