Mass Spectrometry Obaid M. Shaikh

“Mass spectrometry is the branch of science dealing with all aspects of mass spectroscopes and the results obtained with these instruments.’’ 1. It measures mass better than any other technique. 2. It can give information about chemical structures. To identify, verify, and quantify: metabolites, recombinant proteins, proteins isolated from natural sources, oligonucleotides, drug candidates, peptides, synthetic organic chemicals, polymers etc

Applications of Mass Spectrometry Pharmaceutical analysis Bioavailability studies Drug metabolism studies, pharmacokinetics Characterization of potential drugs Drug degradation product analysis Screening of drug candidates Identifying drug targets Bio-molecule characterization Proteins and peptides Oligonucleotides Environmental analysis Pesticides on foods Soil and groundwater contamination Forensic analysis/clinical

Theory: Ionization A gas phase molecule subjected to energy* greater than the ionization energy electron can be removed which results in the formation of a molecular ion:

Working of mass spectrometer Sample Ion source: makes ions Mass analyzer: separates ions Mass spectrum: presents information

Mass Spectrometer Block Diagram High Vacuum System Ion source Mass Analyzer Data System Inlet Detector

Mass Spectrometer Block Diagram Turbo molecular pumps High Vacuum System Ion source Mass Analyzer Data System Inlet Detector

Types of Vacuums Rough High Ultra High Mechanical Pumps oil diffusion/ turbomolecular pump in series with a mechanical pump Ultra High cyropumps or ion pumps in series with mechanical pumps

Sample Introduction High Vacuum System Ion Source Mass Analyzer Data Inlet Detector HPLC Flow injection Sample plate

Ion Source High Vacuum System Ion Source Mass Analyzer Data System Inlet Detector MALDI ESI LSI (Liq. Sec. Ion) FAB EI CI

Ionization Techniques Gas-Phase Methods Electron Impact (EI) Chemical Ionization (CI) Desorption Methods Matrix-Assisted Laser Desorption Ionization (MALDI) Fast Atom Bombardment (FAB) Spray Methods Electrospray (ESI)

Electron Impact Advantages Well-Established Fragmentation Libraries No Supression Insoluble Samples Interface to GC Non-Polar Samples Disadvantages Parent Identification Need Volatile Sample Need Thermal Stability No Interface to LC Low Mass Compounds (<1000 amu) Solids Probe Requires Skilled Operator

Electron Impact

Chemical Ionization Disadvantages Advantages No Fragment Library Parent Ion Interface to GC Insoluble Samples Disadvantages No Fragment Library Need Volatile Sample Need Thermal Stability Quantitation Difficult Low Mass Compounds (<1000 amu) Solids Probe Requires Skilled Operator

MALDI: Matrix Assisted Laser Desorption Ionization Sample plate Laser hn Sample is mixed with matrix (X) and dried on plate. Laser flash ionizes matrix molecules. Sample molecules (M) are ionized by proton transfer: XH+ + M  MH+ + X. MH+ Grid (0 V) +/- 20 kV

FAB: Fast Atom Bombardment Advantages Parent Ion High Mass Compounds (10,000 amu) Thermally Labile Compounds (R.T.) Disadvantages No Fragment Library Solubility in Matrix (MNBA, Glycerol) Quantitation Difficult Needs Highly Skilled Operator Relatively Low Sensitivity

Electrospray ionization Advantages Parent Ion High Mass Compounds (>100,000 amu) Thermally Labile Compounds (<0º C) Easy to Operate Interface to HPLC Zeptomole sensitivity with nanospray Disadvantages No Fragmentation Need Polar Sample Need Solubility in Polar Solvent (MeOH, ACN, H2O, Acetone are best) Sensitive to Salts Supression

Mass Analyzer High Vacuum System Ion source Mass Analyzer Data System Inlet Detector Double Focusing Time of flight (TOF) Quadrupole Ion Trap Magnetic Sector FTMS

Mass analyzers separate ions based on their mass-to-charge ratio (m/z) Operate under high vacuum (keeps ions from bumping into gas molecules) Actually measure mass-to-charge ratio of ions (m/z) Key specifications are resolution, mass measurement accuracy, and sensitivity. Several kinds like, quadrupole, time-of-flight and ion traps etc. are mostly used.

Quadrupole Mass Analyzer Uses a combination of RF and DC voltages to operate as a mass filter. Has four parallel metal rods. Lets one mass pass through at a time. Can scan through all masses or sit at one fixed mass.

Quadrupoles have variable ion transmission modes mass scanning mode m1 m3 m4 m2 single mass transmission mode m2 m3 m1 m4

Time-of-flight (TOF) Mass Analyzer Source Drift region (flight tube) + + detector + + V Ions are formed in pulses. The drift region is field free. Measures the time for ions to reach the detector. Small ions reach the detector before large ones.

Quadrupole Ion Trap Electric/magnetic fields trap, store, eject ions Varying RF frequency will vary the m/z ratios that are trapped Ions may be stored either destructively or non-destructively Additional fragmentation can be performed on ions stored in the ion trap Cut away side view

Detector High Vacuum System Ion source Mass Analyzer Data System Inlet Microchannel Plate Electron Multiplier Hybrid with photomultiplier Faraday Cup

Electron Multiplier

Microchannel plate + e - primary ion L D 1000V 100V L >> D

Data System High Vacuum System Ion source Mass Analyzer Data System Inlet Detector

The mass spectrum shows the results as

Mass spectrometers get their names based upon Types of ion sources: Electrospray (ESI) Matrix Assisted Laser Desorption Ionization (MALDI) Types of mass analyzers: Quadrupole (Quad, Q) Ion Trap Time-of-Flight (TOF) - Either source type can work with either analyzer type: “MALDI-TOF,” “ESI-Quad.” Analyzers can be combined to create “hybrid” instruments. ESI-QQQ, MALDI QQ TOF, Q Trap etc.

Summary: acquiring a mass spectrum Ionization Mass Sorting (filtering) Detection Ion Source Ion Detector Mass Analyzer Form ions (charged molecules) Sort Ions by Mass (m/z) Detect ions 100 75 Inlet • Solid • Liquid • Vapor 50 25 1330 1340 1350 Mass Spectrum

Important Performance Factors Mass accuracy: How accurate is the mass measurement? Resolution: How well separated are the peaks from each other? Sensitivity: How small an amount can be analyzed?

Gas Chromatography/Mass Spectrometry MS is the most dependable technology for forensic chemistry analysis of drugs and toxicological analysis The use of combined gas chromatography-mass spectrometry (GS/MS) is applied in forensic chemistry Gas chromatography helps provide the identification for the presence of a compound of interest Mass spectrometer provides a fragmentation pattern, which can be compared with the referenced sample

MS/MS or Tandem Mass Spectrometry MS/MS means using two mass analyzers (combined in one instrument) to select an analyte (ion) from a mixture, then generate fragments from it to give structural information. Mixture of ions Single ion Fragments Ion source MS-1 MS-2

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