Bioanalytical methodologies with emphasis on use of chromatography and mass spectrometry in metabolomics analysis

Content

Basic chromatographic theory

• The separation and band broadening processes
• Chromatographic terms
• Adapting to rules from regulatory bodies (FDA, EMA, Eur Pharmacopoeia

Introduction to Gas Chromatography (GC)

• Carrier gases
• Application of chromatographic theory in GC
• Injection techniques in GC:
  • Split/Splitless injection
  • (PTV, On Column and Head Space will be discussed in brief)

Columns in GC

• Wall Coated Open Tubular Columns (WCOT)
  • Effect of length
  • Effect of internal diameter
  • Effect of stationary phase thickness
  • Phase Ratio
  • The Retention Index (RI)-system
  • McReynold classification of chemical properties of the stationary phase
• SCOT and PLOT columns will be discussed in brief

Introduction to Mass spectrometry (MS)

• MS- how does it work?
  • Ion source
  • Mass analyser
  • Detector
• The EI- ion source. EI spectra
• MS-instrumentation
  • Mass range
  • Accuracy
  • Resolution
• Obtaining information from EI-MS spectra (I)
  • The use of the natural abundance of stable isotopes of different elements
  • Calculating the number of C atoms in a molecule
• Obtaining information from EI-MS spectra (Ii)
  • The occurrence of Cl, Br and N in a molecule
  • Calculating the elemental composition of a molecule
  • Calculating the number of double bond equivalents (DBE) in a molecule
• Obtaining information from EI-MS spectra (IiI)
  • The use of natural, neutral losses
  • The use of diagnostic m/z-values
• The use of GC-MS to obtain EI-MS spectra
  • The importance of tuning and calibration
  • How to obtain a spectrum from raw data
  • EI spectra and libraries
• GC-MS and quantitative analysis
  • Full Scan, SIR and MRM
  • Adapting to rules from regulatory bodies (WADA, EMA, FDA)
  • Validation

Introduction to Liquid Chromatography – Mass Spectrometry (LC-MS)

• Instrumentation in LC-MS
  • HPLC/UHPLC instrumentation
  • Mobile phases in HPLC
  • Synthesis of Bonded Phase Columns
  • The retention mechanisms of RP-HPLC (C18) columns)
    • Other columns (HILIC, mixed mode etc. will be discussed in brief)
  • Controlling retention time and selectivity
  • Transferring HPLC-UV methods to UHPLC-MS methods
    • Which buffers and additives to mobile phase can be used in LC-MS
• The application of Chromatographic theory in LC
  • The effect of column length
  • The effect of column internal diameter
  • The effect of particle diameter (dp)
  • Finding the optimal flow rate
• Instrumentation for LC-MS
  • The ESI- ion source
  • Ion suppression
  • In Source fragmentation
  • Obtaining ESI spectra for different purposes
  • ESI spectra and libraries
    • Other ion sources (APCI, MALDI will be discussed in brief)
• Interpretation of ESI-LC-MS spectra
• LC-MS quantitative analysis
  • Method development
• Validation of quantitative analysis
  • The design of a validation plan
  • The validation processes
    • (The lecture will adapt to Guidelines from FDA and/or EMA)
• Application of GC-MS and LC-MS
  • The use in bioanalysis (exemplified with Therapeutic Drug Monitoring – TDM)
  • GC-MS and LC-MS in metabolomics
  • LC-MS in proteomics

Dates

The course is divided in 2 modules. The course dates are February 18-22 2019 (module 1) and March 11-15 2019 (module 2).

Place

Umeå Plant Science Centre (UPSC), Umeå University

Link to the registration form

The registration is closed now!

Language

English

Teaching

• Oral lectures
• Group Work
• Discussion of results from group work
• Home work (between module 1 and 2)
• Present results from home work

Examination

In order to be approved, the student must be part of the group work and presentations, and approved homework, oral or written.

Credits

Approved course corresponds to 6 HTC