LC-MS/MS Technique & Optimization Course

"Discover the Whole Power and Exciting Potential of LC-MS/MS"

November 11-12, 2010, Toronto, Canada

About This Course | Course Syllabus | Instructor Biography

Registration Fees | Online Registration | Travel and Accomodation

Course Syllabus

LC-MS/MS Technique

LC-MS Inlets: Coupling HPLC with a Mass Spectrometer
- Introduction to Mass Spectrometry
  - What is a Mass Spectrometer?
  - Mass Spectrum
- Main components of a Mass Spectrometer
- GC and HPLC coupled to MS: GC-MS vs. LC-MS
- HPLC-UV versus LC-MS
- Arpino's Symbolism
- Interfaces and flow-rates
- Brief historical background on LC-MS inlets:
  - Stopped flow
  - Disk interface
  - Particle Beam
  - Thermo Spray
  - Moving Belt

Ionization Techniques
- Ion production and fragmentation
  - Hard vs. soft ionization
- Electron Impact (EI) Ionization
- Chemical Ionization (CI)
- EI and CI for GC-MS
  - Capillary direct inlet
  - Split inlets
  - Jet Separator
- Direct Inlet Probe (DIP)
  - DIP-EI and DIP-CI
- Fast atom bombardment (FAB)

API and MALDI Sources
- Introduction to Matrix-assisted laser desorption/ionization (MALDI)
  - Common matrix in MALDI
- Atmospheric Pressure Ionization (API)
  - Analytical domains
- Electrospray (ESI)
  - Introduction to Ion Evaporation
  - Strengths and weaknesses
  - Heated capillary (Thermo) vs. curtain gas (Sciex)
  - Agilent and Waters approches
  - Ionspray (IS): pneumatically assisted ESI
  - Turboionspray (TIS): effects of the drying gas
  - Formation of adducts and clusters
  - Introduction to the good/bad modifiers
  - Needle voltage, nebulizer flow and background information on solvent composition
- Atmospheric Pressure Chemical Ionization (APCI)
  - Gas phase proton transfer
  - Corona Discharge
  - Practical considerations on buffers, probe temperature and solvent composition
  - Ion formation in APCI and the 3 steps process
  - Importance of proton hydrates in APCI
  - Heated Nebulizer (HN)
- Advantages and disadvantages of soft ionization sources
- Contaminants

Mass Analyzers
- How mass analyzers work: Mass filter
- Brief introduction to Magnetic Sector
  - Single focusing and double focusing
- Ion trap
  - Tandem in Time vs. Tandem in Space
  - The 4 steps of ion trap: Collection, isolation, excitation and ejection
  - Basic ion trap components
  - Space charging
  - 3D traps vs. Linear traps
- Time of Flight (TOF)
  - Drift region
  - Spatial spread
  - Reflector
  - TOF analyzer process
  - TOF data acquisition
- Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS or FT-MS)
- What are the advantages and disadvantages of various mass analyzers in applications
  - m/s range vs. Resolution vs. Accuracy
  - Single Quad vs. Triple Quad vs. Ion Trap vs. TOF

Quadrupole
- Schematic diagram of a quadrupole mass spectrometer
- RF in a quadrupole
- Quadrupole Theory
- Types of voltages present on a quadrupole: RF and DC
  - What is RF?
- "RF only" mode
- Ion trajectories
- Ion Stability
- Quadrupole settings
- Mathieu Equation and stability diagram
- Profile scan
- Bandwidth and peak width

Single Quadrupole and Triple Quadrupole Operations
- MS calibration
  - Peak width
  - Peak shapes
  - Peak assignments
  - Resolution vs. sensitivity
  - Scan line
- Single Quadrupole (MS) Operations:
  - Full Scan
  - Selected Ion Monitoring (SIM)
- Tandem Mass Spectrometry (MS/MS)
  - Fragmentation and why is important
  - Collision Cell and CID
- Understanding Triple Quadrupole (QqQ) Operations:
  - Precursor Ion Scan
  - Multiple or Selected Reaction Monitoring (MRM or SRM)
  - Product Ion Scan
  - SIM vs. SRM:
    - A case study: Racehorse drug testing

Ion Path: Ion Transmission
- Declustering Region
- Lents and ion focusing
- Skimmer
- Mass filter rail
- Ion Detection:
  - The detector
  - Electron multiplier optimization

LC-MS/MS Optimization

APCI Optimization
- Importance of discharge needle position
- APCI main components in Sciex, Thermo, Waters and Agilent sources
- Gases optimization
- Probe Temperature and flow rate
- Solvents

APPI Optimization
- Theory and applications of atmospheric pressure photoionization (APPI) interface
  - Principles of photoionization
  - Direct photoionization
  - Selection ionization: Ionization potentials
- Bruins & Robb's approach
- Syage & Hanold's approach
- Electron and proton transfer
  - The dopant and ionization efficiency in APPI
  - Common dopants
- When APPI really works well:
  - Naphthalene & diphenyl sulfide vs. carbamazepine & acridine

ESI Optimization
- Problems with On axis spray
- Optimization of sheath/nebulizer gas and auxiliary/make up gas
- Use of Make up liquid for optimizing the ionization
- ESI main components in Sciex, Thermo, Waters and Agilent sources
- Common problems with ESI
  - Signal instability
  - Arching
- Advantages of Orthogonal spray
- The V.I.C. Rule: Volatility, Ion Paring and Conductivity
- ESI Additives & Buffers to use for the best sensitivity
  - Proton donors
  - Proton acceptors
  - Buffers
  - Ion pairing reagents: compromising sensitivity vs. chromatographic resolution
  - Ion pairing efficiency
    - Phosphate, sulfate and borate buffers
  - Negative ion formation
  - Compatibility with HPLC technique
- The infusion experiment to enhance the sensitivity in ESI
  - Experiment design: HPLC pumps + Infusion pump
  - Solvents preparation: 50/50 MeOH/Water vs. 50/50 ACN/Water
  - Modifiers preparation: 1% Acetic acid; 0.1% formic acid; 5-10mM Ammonium acetate; 0.1-0.02% TFA; 0.1% Ammonium hydroxide
  - Case study: Achieving the best sensitivity for a pentapeptide
- The 5 Golden Practical rules in ESI
  - Final considerations and strategies on LC mobile phases
- Tricks and tips of using divert valve with isocratic/gradient chromatography to enhance sensitivity
- Working in the highest sensitivity conditions: "Is it possible?"

Knowing ESI Theory to improve source Optimization
- A message from Prof. Fenn (Nobel prize for Chemistry in 2002)
- ESI Definition and Advantages
- The 4 major processes in ESI
- Ion evaporation: surface tension and droplet production
- D&D: Desorption & Desolvation
- Rayleigh Limit
- Practical considerations from ESI Theory
  - ESI mechanism for small molecules
  - Hydrophilic vs. hydrophobic molecules
  - Case study: Tetra alkyl ammonium halides
  - ESI mechanism for large molecules and Proteins
    - Charged residue mechanism (CRM)
  - Droplet vs. protein
- Dole/Fenn theory vs. Iribarne theory
- ESI as Electrochemical Cell
  - Platinum ESI capillary vs. stainless steel ESI capillary
  - The electrochemical effect

Fundamentals of Protein Analysis and Nanospray Optimization
- Functional and structural proteins
- Basic understanding of multiple charging of proteins
- Transcription and translation
- Primary sequence and amino acids that produce positive and negative charges
- MS/MS nomenclature
- Approaches to data analysis for protein identification and characterization
  - Fingerprinting and its limitations
  - Tag
  - De novo
  - Post translation modifications
  - Case study: Identification of possible phosphorylation sites
- Protein identification and sequencing with MALDI-MS/MS and LC-ESI-MS/MS
- Multiply changed ions
  - Calculations for the charge state
  - Case study: Angiotensin
  - Case study: Interleukin
- Nanospray
  - Disposable tips
  - Tips loading and positioning
  - Chromatography in the tip
  - Tandem LC - Tandem MS (LC/LC-MS/MS)
- Proteomic case studies:
  - Fibrinopepide
    - Charge state (MS)
    - Identification: Tag in MS/MS
  - Myoglobin
    - Sample Digestion
    - Charge state (MS)
    - Identification: Tag in MS/MS

MicroESI Optimization
- Routine capillary analysis
- Main advantages of using microESI
  - High sensitivity and low injection volume
  - Sharp chromatographic peaks
  - Extremely low flow rate and chemical noise
- Columns for microESI
  - Flow rate & column optimization
- Why does microESI work so well?
- **Acetonitrile (ACN) Worldwide shortage (2009 Special lecture) & MicroESI**

Ion Transmission Optimization
- Inefficiency of the ionization process
- How and where to improve ion transmission efficiency in triple quads and ion traps
- Orifice - Ion focusing - Skimmer transfer: Sciex vs. Thermo
- Importance of the vacuum system
- Multipoles characteristics:
  - Round quadrupole
  - Octapole
  - Square quadrupole
- Collisional focusing & cooling
  - Advantages and disadvantages
- Collision induced dissociation (CID) efficiency
- Source CID
  - Main applications

Crosstalk and Collision Cell
- "Is your instrument really Crosstalk free?"
- "What is crosstalk?"
- High pressure collision cell
- LINAC (Sciex), Twave (Waters), Hexapole Axial Acceleration (Agilent), and Curved Collision Cells (Thermo & Varian)
- Dwell time and Crosstalk
- Crosstalk and its impact on:
  - Selected Reaction Monitoring (SRM): dwell period and inter-channel delay
  - Precursor ion scan (PIS)
  - Neutral loss scan (NLS)
- Case studies
- Forensic toxicology case study: Amphetamine analysis

Optimization software
- MS parameters
- Resolution
- MS/MS auto optimization

Advantages of LC-MS/MS over LC-MS

New and emerging LC-MS technologies