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Lcms Basics
- 1. Long Island University| April 6th, 2018 LC-MS/MS Basics Ben Kuzma
- 2. Long Island University| April 6th, 2018 Objectives • Introduce basic MS concepts • Explain the fundamentals of MS • Demonstrate the benefits of different systems • Method Development using concepts
- 3. Long Island University| April 6th, 2018 Key Terms • Parent Ion (Precursor ion) • Daughter Ion (fragment ion) • MS/MS • MRM http://www.waters.com/webassets/cms/library/docs/720000807en.pdf
- 4. Long Island University| April 6th, 2018 Overview https://www.idex-hs.com/education-and-tools/educational-materials/hplc-center
- 5. Long Island University| April 6th, 2018 Overview https://www.idex-hs.com/education-and-tools/educational-materials/hplc-center min 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 % 0 100 F1:MRM of 2 channels,ES+ 172 > 127.9 Metronidazole_400pg_ml_Lot_1_2 Smooth(Mn,1x2) Metronidazole 400pg/ml 1.221e+005Metronidazole 1.54 5460.39 116590 1.11 2.131.97 1.89 2.58 2.79
- 6. Long Island University| April 6th, 2018 Why use LCMS? • Selectivity • Lidocaine and Prilocaine • Elute with 0.1 minutes of each other • Speed (UHPLC) • HPLC Run time – 5-20 minutes • UHPLC Run time 3 minutes • Sensitivity • HPLC LLOQ - 10 ng/ml • LCMS/MS – 400 pg/ml (0.4ng/ml) • 25 fold difference
- 7. Long Island University| April 6th, 2018 Fundamentals of LCMS • How does it work? • Mass to charge ratio (m/z) • Ionization Methods • Electrospray ionization (ESI) • Atmospheric Pressure Chemical ionization (APCI) • Atmospheric pressure photoionization (APPI)
- 8. Long Island University| April 6th, 2018 Fundamentals of LCMS Liquid Chromatography Ion Source Mass Spec Detector/waste http://www.ecs.umass.edu/eve/background/methods/chemical/Openlit/Chromacademy%20LCMS%20Intro.pdf
- 9. Long Island University| April 6th, 2018 Ionization Methods • Electrospray ionization (ESI) • Limited fragmentation http://www.particlesciences.com/news/technical- briefs/2009/mass-spectrometry-bioanalysis.html
- 10. Long Island University| April 6th, 2018 Ionization Methods • Atmospheric Pressure Chemical ionization (APCI) http://www.particlesciences.com/news/technical- briefs/2009/mass-spectrometry-bioanalysis.html
- 11. Long Island University| April 6th, 2018 Ionization Methods • Atmospheric pressure photoionization (APPI) http://ccc.chem.pitt.edu/wipf/Agilent%20LC-MS%20primer.pdf
- 12. Long Island University| April 6th, 2018 Type of Mass Detectors • Quadrupole • Ion Trap • Time of Flight • Magnetic Sector Triple Quad = 2 Mass analyzers
- 13. Long Island University| April 6th, 2018 Quadrupole • Uses electric field to separate ions of interest (volts) • If the m/z ratio is “correct” then it will not collide with the poles and it will continue on to the next part of the analyzer. • Gold standard for quantitation (QqQ)
- 14. Long Island University| April 6th, 2018 Ion Trap • Trap the ions of interest in a field then release them m/z intensity
- 15. Long Island University| April 6th, 2018 Time of Flight (TOF) • Fast Scan rate • Metabolites • Structural elucidation m/z intensity F Ion Source Acceleration Time - of - Flight Detector
- 16. Long Island University| April 6th, 2018 LC-MS/MS • Gold standard for quantitation http://what-when-how.com/proteomics/quadrupole-mass- analyzers-theoretical-and-practical-considerations- proteomics/
- 17. Long Island University| April 6th, 2018 Application of Fundamentals
- 18. Long Island University| April 6th, 2018 Ionization type • ESI • Positive Mode • Negative Mode • APCI Waters LCMS Prep Guide
- 19. Long Island University| April 6th, 2018 MS Scan Mass Range of 120 – 600 m/z
- 20. Long Island University| April 6th, 2018 MS Scan
- 21. Long Island University| April 6th, 2018 ESI+ 120-600 M/z
- 22. Long Island University| April 6th, 2018 Daughter Ions Argon Gas Also called MSMS Mode
- 23. Long Island University| April 6th, 2018 Daughter Ions
- 24. Long Island University| April 6th, 2018 Daughter Ions
- 25. Long Island University| April 6th, 2018 Daughter Ions Argon Gas
- 26. Long Island University| April 6th, 2018 10 eV 15 eV 20 eV 25 eV 30 eV
- 27. Long Island University| April 6th, 2018 Daughter Ions Argon Gas
- 28. Long Island University| April 6th, 2018 Development of MRM MW +H Transition (MSMS) Time transitions are analyzed Voltage applied during ionization Energy of Argon gas for fragments
- 29. Long Island University| April 6th, 2018 Final Product of MRM
- 30.
- 31. Resources • http://ccc.chem.pitt.edu/wipf/Waters%20LC-MS%20primer.pdf • http://www.ecs.umass.edu/eve/background/methods/chemical/Ope nlit/Chromacademy%20LCMS%20Intro.pdf •http://www.rsc.org/images/AMC%20LCMS%20Guide_tcm18- 240030.pdf
Editor's Notes
- #5 LC-MS/MS – What is it? Liquid Chromatography (HPLC) MS(/MS) – simply a different detector
- #6 LC-MS/MS – What is it? Liquid Chromatography (HPLC) MS(/MS) – simply a different detector
- #7 Disadvantages Complexity Cost Excesive selectivity
- #8 Can be in positive mode (M+) or in negative mode (M-)
- #9 Disadvantages Complexity Cost Excesive selectivity
- #10 The LC eluent flows through a metal capillary contained within the probe. Droplets are formed by nebulisation of the LC flow into a spray as it leaves the electrospray capillary. A charge is transferred onto the droplets by applying a large (2-5 kV) potential difference between the electrospray capillary and counter electrode. The droplet size reduces by evaporating the mobile phase by the use of a heated drying gas. This desolvation increases charge density on the surface of the smaller droplets. Electric repulsion due to the charge density results in droplet fission. When this exceeds the surface tension of the droplet it results in coulombic fission. Gas-phase ions are formed as the droplet “explodes” and are sampled typically through some form of orifice. Ionization occurs in the mobile phase
- #11 The LC eluent flows through a silica capillary contained within the probe. Droplets are produced by nebulisation of the LC flow into a spray. Solvent is evaporated by the use of a heater in the probe to produce gas-phase molecules. A corona discharge needle placed in the ion source generates electrons and ionises air and gaseous solvent molecules forming reactive species (N2+, H2O+, O2+), which quickly react to form H3O+(H2O)n. Proton transfer from/to the ionised solvent results in chemical ionisation of analyte [M+H]+. Ionization takes place in the source
- #12 Accomplished by exposing a spray of droplets to photoirradiation The ion is formed when the molecule absorbs a photon Not commonly used
- #13 Quadrupole – 4 poles that guide the specified ion through Ion Trap – traps the ion of interest and multiplies it Time of flight – the heavy the molecule the less it will travel Magnetic sector – a magnetic beam guides the ions to the detector
- #15 Ions are held by a certain frequency and then a ramp is applied so that ions of a m/z come out at a certain point and then the next set of ions are pushed out
- #16 The ionize are accelerated and the ionzed that are lighter will reach the detector first and ones that are heavier will reach later (according to m/z)
- #17 Collison gas is argon Atmospheric pressure ionization gas is nitrogen Two sets of quadrupoles
- #19 Just an example of the ionization types and positive or negative mode
- #23 Shows the ions of a certain mass range going through the first quad The ions are then hit with a collison gas – argon at different voltages
- #25 Changed the gradient Centroid data is what I showed before with the peaks – zero width and an exact m/z Profile is the other option that you can have that gives you UV type peaks which makes it difficult to select a transition
- #26 Shows the ions of a certain mass range going through the first quad The ions are then hit with a collison gas – argon at different voltages
- #28 Shows the ions of a certain mass range going through the first quad The ions are then hit with a collision gas – argon at different voltages
- #29 Shows the ions of a certain mass range going through the first quad The ions are then hit with a collision gas – argon at different voltages
- #30 Shows the ions of a certain mass range going through the first quad The ions are then hit with a collision gas – argon at different voltages