Absolute quantitation of small molecules in various matrixes.
- Selected Reaction Monitoring (SRM) based targeted quantitative analysis
- Typical project workflow
- Getting started – small molecules
- Standard assays - the following assays have been partially validated for specific biological matrices
- Technical principles – Selected Reaction Monitoring (SRM) scanning mode allows highly sensitive and selective analyte detection by separating the ions of interest unique to the analyte from those of the complex biological matrix
- Instrumentation – the most common instrument used for quantitation is a triple quadrupole mass spectrometer coupled with a liquid (LC) or gas chromatography (GC) system. Depending on the nature of the analyte, the mass spectrometer ionization technique may be electrospray (ESI), atmospheric pressure chemical ionization (APCI) or electron ionization (EI).
- Sample preparation – the sample preparation approach depends vastly on the physicochemical properties of the analyte and the type of sample matrix. Most common approaches include dilute-and-shoot, protein precipitation, liquid-liquid extraction, and solid phase extraction (SPE)
- Cost estimate
- More information here: 2008 Stanford Mass Spectrometry Users' Meeting
- MS method development – direct MS and MS/MS analysis of the analyte, optimization of MS parameters
- LC method development – determination of appropriate column and buffer system to achieve optimal separation in conjunction with analysis length
- LC-MS/MS method optimization and basic validation – determination of limits of detection (LOD) and linearity range for the calibration curve; assessment of potential matrix interferences and stability of the analyte (if necessary)
- Sample preparation – determination of the most practical sample preparation procedure. Goals are efficient sample clean up with the highest extraction efficiency and minimal sample handling
- Sample analysis - analysis of samples using the above established LC-MS/MS method
- Authentic standard for the analyte – can be purchased from many companies
- Internal Standard (IS) – the ideal internal standard is a stable isotope labeled version of the analyte molecule (13C, 2H, 15N) which is at least 3Da higher in mass than the analyte itself. Alternatively, a structural isomer or chemical analog of the analyte can be used. Labeled standards are offered by numerous companies.
- Blank matrix – availability of analyte-free matrix is crucial to method development. In select cases, matrix with endogenous levels of the analyte may be used.
- Consultation required – Quantitative assays are custom projects and require in-depth discussion of the project and experimental design before the samples are generated. If you are interested in running a quantitative LC-MS/MS assay, please contact SUMS staff to set up a meeting. Please contact Karolina Krasinska, Ludmila Alexandrova or Allis Chien