LC-MS/MS

TSQ (Triple Quadrupole Mass Spectrometry)

https://www.enovatia.com/wp-content/uploads/2009/07/Scan_types.pdf
https://www.enovatia.com/wp-content/uploads/2009/07/Scan_types.pdf
  • Quadrupoles are mass analyzers(mass filters) which consist of four rods with DC and RF voltages applied. An ion of a specific (m/z) ratio will be stable and can pass through the quadrupole only when a specific DC/RF voltage combination is applied.
  • Linear ion trap (LIT) is a type of ion trap mass spectrometer. In a linear ion trap, ions are confined radially by a two-dimensional radio frequency (RF) field, and axially by stopping potentials applied to end electrodes. Linear ion traps have high injection efficiencies and high ion storage capacities.

Product ion scans/Daughter ion scans

  • Q1 is set to allow only the transmission of one m/z.
  • Product ions are scanned through Q3.
  • There are two types of product ion scans :
  • 1. Full Scan Product ion are used for qualitative applications to obtain structural information.
  • 2. SRM (Selected Reaction Monitoring) Product ion scans are used for Quantitative target analysis.
  • LC-MS/MS run-times are much faster than GC-MS and LC-UV.
  • Sample Preparation Techniques
    • Dilute ‘n Shoot
    • Protein Precipitation
    • Liquid-Liquid Extraction
    • Solid Phase Extraction
  • SIM Vs MRM– In MRM typically a single signal per analyte is detected while SIM can result in multiple signals per compound.

Instruement Parameters

https://sciex.com/Documents/Downloads/Literature/turbo-v-operator-guide-en.pdf
  • SOURCES/GAS PARAMATERS
  • depend on LC conditions , should be optimized near desired LC flow conditions.
    1. IS
    2. TEM
    3. CUR
    4. GS1
    5. GS2
https://www.uab.edu/proteomics/metabolomics/workshop/2014/Ray%202014%20MRM%20Development%20Demo.pdf
  • IonSpray Voltage (IS)
    • Controls the voltage applied to the needle/electrodes/sprayer in the ESI probe, which ionizes the sample in the ion source (not in APCI probe).
    • Optimum IS value depends on depends on the polarity and flow rate (IS value decreases with flow rate (5 ul-3 ml/min)).
    • it affects the stability and the sensitivity of the spray.
  • Temperature (TEM)
    • Controls the temperature of (turbo gas in TurboIonSpray) heat applied to the sample to vaporize it.
    • Optimal temperature is the lowest temperature at which the sample is vaporized completely.
    • Optimize in increments of 50°C (upto 750 degree C).
  • Ion Source Gas 1 (GS1) (nebulizer gas)
  • Ion Source Gas 2 (GS2) (Heating or drying Gas)
  • Curtain Gas (CUR)
    • CUR flow is introduced into the interface of curtain plate and orifice plate. Curtain plates attract the ions (supplies attractivee action potential)produced by ion source and concentrates them into the region of sampling orifice.
    • Counter-flow of high purity hot nitrogen gas which encloses the ESI spray like a curtain that prevents netral molecules, solvent droplets and solid particles entering the instrument and contamination the ion optics.
    • It accelerates the evaporation processes and significantly increases the sensitivity during ionization.
    • Should keep CUR as high as possible without losing sensitivity (until signal begins to decrease, not below 20).
  • COMPOUND DEPENDENT PARAMETERS
    1. DP
    2. EP
    3. CE
    4. CES
    5. CXP

  • Declustering potential (DP) is a voltage applied to the orifice that helps to prevent the ions from clustering together and minimize the solvent cluster ions (maximize analyte entry) into MS.
  • Entrance potential(EP) is a voltage applied to the first set of quadrupole (Q0) to guide and focus ions into MS.
  • Collision energy (CE) is the energy that precursor ion receives when they get accelerated and get fragmented by collision with CAD gas molecules inside collison cell Q2.Collisional Energy is converted into vibrational energy, and bonds break.
  • It’s the potential difference between Q0 and Q2 for MS/MS scans.
  • Collision energy spread (CES)
    • During filling of ion-trap this collision energy is stepped (ramped) across a range of energies. Collision energy (CE) represents the center value and CES tells about the energy values on either sides. For ex-if CE=30v and CES=5V. Ion trap fill time will split into 3 durations and each of the one third part of this time uses 25, 30 and 35V collision energy.
  • Collision Cell Exit Potential (CXP)-it focuses and accelerates the ions out of the collison cell Q2 into Q3.
https://www.uab.edu/proteomics/metabolomics/workshop/2014/Ray%202014%20MRM%20Development%20Demo.pdf
https://www.uab.edu/proteomics/metabolomics/workshop/2014/Ray%202014%20MRM%20Development%20Demo.pdf
https://www.uab.edu/proteomics/metabolomics/workshop/2014/Ray%202014%20MRM%20Development%20Demo.pdf

Selectivity vs specificity of a method

  • Selectivity refers to the ability of the method to discriminate a particular analyte in a complex mixture without interference from other components. In the other hand, specificity can be considered as the ultimate selectivity, i.e. 100% selectivity (or 0% interferences).

Interface/Ionization source in MS

ESI normally requires pre-protonated ions in solution and thus works best with fairly basic (or acidic) compounds, whereas APCI is also well suited for the analysis of more neutral compounds.

  • Atmospheric Pressure Ionization (API), a type of soft ionization technique, which ionizes compounds under atmospheric pressure conditions making it especially useful for removing solvents outside a vacuum.
  • API serves as both the ionization source and the interface in a LC-MS system. It facilitates the transition from a high pressure (flowing eluate) environment (HPLC) to high vacuum conditions needed at the MS analyzer.
  • In general, ions generated by API are stripped of solvent, focused into a beam using an ion guide, and finally introduced into the mass analyzer.
  • Three API techniques are, 1. electrospray ionization (ESI), 2.atmospheric pressure chemical ionization (APCI) and 3.atmospheric pressure photoionization (APPI).
  • They can also be commercially available as dual ESI/APCI, ESI/APPI, or APCI/APPI ion sources.
https://www.shimadzu.com/an/lcms/support/fundamental/interfaces_for_lcms.html#:~:text=API%20serves%20as%20both%20the,introduced%20into%20the%20mass%20analyzer.
Electrospray ionization (ESI)
  • This ion source/ interface can be used for the analysis of moderately polar molecules (e.g., metabolites, xenobiotics, and peptides).
  • Liquid phase ionization takes place in ESI.
  • Ideal for selection of precursor ions and for maximizing sensitivity due to little fragmentation of protonated or de-protonated molecules.
  • “Matrix effect”due to presence of co-eluting analytes or co-eluting matrix components is major sources of uncertainty.
  • Response can be non-linear at high concentrations and optimum pH for ESI response can conflict with choices made to control LC selectivity.
  • The solvent should have following properties:
  • have some dipole moment to support ions in solution.
  • less viscous and volatile for better sensitivity.
  • higher % of organic modifier gives better sensitivity due to the decreased surface tension and lower solvation energies for polar analytes.
  • Reversed phase solvents are suitable as they are often polar.
Atmospheric pressure chemical ionization (APCI)
  • APCI ion source/ interface can be used to analyze small, neutral, relatively non-polar, and thermally stable molecules (e.g., steroids, lipids, and fat soluble vitamins). These compounds are not well ionized using ESI.
  • In addition, APCI can also handle mobile phases containing buffering agents.
  • Given that APCI is a gas-phase ionisation technique ion-suppression is not as much as ESI.
  • APCI needs greater concentrations of additives in the mobile phase to control the chemical ionization process taking place in the gas-phase. Hence, considerable care is needed when selecting the type of solvent and additives used for the mobile phase to avoid issues with suppression of the analyte signal from preferential ionization of solvents or additives with relatively higher proton affinities.
  • Corona Discharge (CD) is a widely studied and applied electrical phenomenon where a gas surrounding a high voltage electrode forms an ionized gaseous plasma.
  • Most solvents are compatible.
Atmospheric pressure photo-ionization (APPI)
  • APPI interface is similar to the APCI ion source, but instead of a corona discharge, the ionization occurs by using photons coming from a discharge lamp.
Additives in LCMS
  • Ion-pairing reagents consist of large ionic molecules having a charge opposite to the analyte and has a substantial hydrophobic chain that allows interacting with the stationary phase, plus associated counter-ions.
  • Use of ion pair reagents can enhance peak shape and retention time when common remedies such as modifying eluent ratios or changing stationary phase fail.
  • The most commonly utilized ion-pair reagents are formic acid (0.05 to 0.2%), and acetic acids (0.3%).
  • TFA (trifluoroacetic acid) (<0.05%) will certainly suppress ionization.
  • Heptafluorobutyric acid (0.05%) , perfluorobutanoic acid and perfluoropropionic acidwhy if formic or acetic aren’t useful.
  • Non-volatile additives will crystallize and block the ion source (e.g. surfactants-triton, SDS).
  • DMSO, TRIS, glycerol can reduce ionization.
  • Corrosive reagents such as inorganic acids (H2SO4, H3PO4) or alkali metal bases (NaOH, KOH) will damage equipment.
  • Strong ion pair reagents (TFA)will cause ion suppression.
  • Additives such as triethyl- and trimethylamine may enhance ion formation in negative mode.

REFERENCES