FAB and MALDI techniques MCQs With Answer

Introduction

This blog provides a focused set of multiple-choice questions on FAB (Fast Atom Bombardment) and MALDI (Matrix-Assisted Laser Desorption/Ionization) tailored for M.Pharm students studying Advanced Instrumental Analysis (MPA 201T). The questions cover fundamental principles, matrices, ionization mechanisms, instrumentation aspects such as coupling with TOF analyzers, sample preparation strategies, advantages and limitations, and pharmaceutical applications including peptide, protein and lipid analysis. Each MCQ is designed to deepen conceptual understanding and exam readiness by addressing practical considerations like matrix selection, salt tolerance, metastable decay, and quantitation challenges. Use these MCQs to test knowledge, identify gaps, and reinforce techniques critical for mass spectrometric analysis in pharmaceutical research.

Q1. What is the primary conceptual difference between FAB and MALDI ionization techniques?

• FAB uses a liquid matrix and atom bombardment to sputter and ionize analytes while MALDI uses a laser-absorbing crystalline matrix to desorb/ionize analytes.
• FAB uses electron ionization in gas phase while MALDI uses chemical ionization.
• FAB requires pulsed lasers whereas MALDI uses continuous ion beams.
• FAB only produces negative ions while MALDI only produces positive ions.

Correct Answer: FAB uses a liquid matrix and atom bombardment to sputter and ionize analytes while MALDI uses a laser-absorbing crystalline matrix to desorb/ionize analytes.

Q2. Which matrix combination is most commonly used for peptide analysis in MALDI?

• α-Cyano-4-hydroxycinnamic acid (CHCA)
• Glycerol
• Cesium chloride
• Perfluorinated oils

Correct Answer: α-Cyano-4-hydroxycinnamic acid (CHCA)

Q3. What is a major advantage of MALDI compared to FAB for high molecular weight biomolecules?

• MALDI produces intact molecular ions with minimal fragmentation and supports much higher mass ranges (proteins and large peptides).
• MALDI requires no vacuum and can be run at ambient pressure.
• MALDI exclusively generates radical cations making interpretation easier.
• MALDI does not require a matrix for ionization of large biomolecules.

Correct Answer: MALDI produces intact molecular ions with minimal fragmentation and supports much higher mass ranges (proteins and large peptides).

Q4. In FAB, what constitutes the primary bombarding species that causes desorption and ionization?

• Neutral fast atoms (e.g., Xe, Ar) generated by an ion source and neutralized before impact
• High-energy photons from a UV laser
• Electrons from a filament producing electron impact ionization
• Positively charged molecular ions

Correct Answer: Neutral fast atoms (e.g., Xe, Ar) generated by an ion source and neutralized before impact

Q5. For which class of compounds was FAB historically most useful in pharmaceutical analysis?

• Low-to-medium molecular weight, polar and thermally labile compounds such as peptides, steroids and drug metabolites.
• Highly volatile small gases like CO and N₂.
• Inorganic salts and metals exclusively.
• High molecular weight intact genomes without fragmentation.

Correct Answer: Low-to-medium molecular weight, polar and thermally labile compounds such as peptides, steroids and drug metabolites.

Q6. Why is MALDI most commonly coupled with a Time-of-Flight (TOF) mass analyzer?

• Because MALDI generates pulsed ion packets that are ideal for TOF’s pulsed flight-time measurement and TOF offers broad mass range and fast analysis.
• Because MALDI ions are charged too slowly for quadrupole analyzers.
• Because TOF is the only analyzer capable of measuring negative ions.
• Because TOF does not require vacuum whereas other analyzers do.

Correct Answer: Because MALDI generates pulsed ion packets that are ideal for TOF’s pulsed flight-time measurement and TOF offers broad mass range and fast analysis.

Q7. What is the primary role of the organic matrix in MALDI?

• To absorb laser energy and mediate desorption/ionization by transferring protons or electrons to the analyte while minimizing analyte fragmentation.
• To chemically derivatize the analyte by covalent bonding.
• To act as a chromatographic stationary phase separating components before ionization.
• To increase the vacuum level in the ion source.

Correct Answer: To absorb laser energy and mediate desorption/ionization by transferring protons or electrons to the analyte while minimizing analyte fragmentation.

Q8. Which limitation is commonly associated with FAB compared to MALDI?

• High background chemical noise from liquid matrices and poor performance for very high molecular weight species.
• Inability to ionize polar molecules.
• Exclusive suitability for imaging tissue sections only.
• Requirement of ultrapure crystalline matrices for operation.

Correct Answer: High background chemical noise from liquid matrices and poor performance for very high molecular weight species.

Q9. Post-source decay (PSD) used for sequencing in MALDI-TOF refers to:

• Fragmentation of ions after desorption in the field-free region, enabling structural/sequence information from intact precursor ions.
• Thermal degradation of the matrix prior to ionization.
• Neutralization of ions on the target plate preventing fragmentation.
• Loss of low-mass ions due to detector saturation only.

Correct Answer: Fragmentation of ions after desorption in the field-free region, enabling structural/sequence information from intact precursor ions.

Q10. Which laser wavelength is classically used in MALDI instruments for matrix excitation?

• UV at about 337 nm (nitrogen laser) or 355 nm (Nd:YAG third harmonic)
• Far-infrared at 10.6 µm (CO₂ laser)
• Green visible laser at 532 nm exclusively
• Microwave radiation at GHz frequencies

Correct Answer: UV at about 337 nm (nitrogen laser) or 355 nm (Nd:YAG third harmonic)

Q11. The predominant ionization mechanism in MALDI for peptides and many small biomolecules is:

• Protonation (formation of [M+H]+) mediated by matrix-to-analyte proton transfer.
• Direct electron impact causing radical cation formation only.
• Cationization exclusively with sodium without proton transfer.
• Thermal vaporization without charge transfer.

Correct Answer: Protonation (formation of [M+H]+) mediated by matrix-to-analyte proton transfer.

Q12. With respect to tolerance to salts and contaminants, MALDI is generally:

• More tolerant of salts than electrospray ionization (ESI), but high salt levels still suppress signal and complicate spectra.
• Completely immune to salt suppression and requires no sample cleanup.
• Less tolerant of salts than ESI and fails with trace amounts of salts.
• Only tolerant to organic contaminants but not inorganic salts.

Correct Answer: More tolerant of salts than electrospray ionization (ESI), but high salt levels still suppress signal and complicate spectra.

Q13. In MALDI sample preparation, the term “sweet spot” refers to:

• A localized region on the co-crystallized target that yields optimum ion signal due to favorable analyte/matrix distribution.
• The point on the laser where energy is minimum to avoid fragmentation.
• A calibration marker used to adjust the mass axis.
• A chemical reagent added to improve ionization efficiency.

Correct Answer: A localized region on the co-crystallized target that yields optimum ion signal due to favorable analyte/matrix distribution.

Q14. Desorption and ionization in FAB primarily result from which process?

• Sputtering by energetic neutral atoms causing ejection of matrix and analyte molecules and subsequent ion formation.
• Ablation by laser pulses absorbed by a crystalline matrix.
• Electrochemical oxidation at the target surface.
• Thermal vaporization induced by resistive heating.

Correct Answer: Sputtering by energetic neutral atoms causing ejection of matrix and analyte molecules and subsequent ion formation.

Q15. Which type of analyte often requires chemical derivatization before MALDI analysis to improve detection?

• Small nonpolar volatile compounds that do not efficiently protonate or cationize.
• Large intact proteins above 100 kDa.
• Peptides with basic residues that ionize readily.
• Inorganic metal ions.

Correct Answer: Small nonpolar volatile compounds that do not efficiently protonate or cationize.

Q16. MALDI imaging (MSI) is most useful for:

• Mapping spatial distribution of drugs, metabolites, lipids and peptides directly on tissue sections.
• Measuring gas-phase reaction kinetics in real time.
• Separating enantiomers in chiral chromatography.
• Determining absolute crystal structure by X-ray diffraction.

Correct Answer: Mapping spatial distribution of drugs, metabolites, lipids and peptides directly on tissue sections.

Q17. Which matrix is commonly preferred for glycan and lipid analysis in MALDI?

• 2,5-Dihydroxybenzoic acid (DHB)
• Glycerol
• Cesium iodide
• Silicon oil

Correct Answer: 2,5-Dihydroxybenzoic acid (DHB)

Q18. Why is a high vacuum required in MALDI-TOF instruments?

• To allow free flight of ions with minimal collisions so accurate time-of-flight separation and high sensitivity are maintained.
• To prevent the matrix from subliming during analysis.
• To enable the laser to function at the correct wavelength.
• To cool the detector electronics for noise reduction.

Correct Answer: To allow free flight of ions with minimal collisions so accurate time-of-flight separation and high sensitivity are maintained.

Q19. Incorporation of a reflectron in a MALDI-TOF analyzer primarily improves which performance metric?

• Mass resolution and mass accuracy by correcting for kinetic energy spread of ions.
• Ionization efficiency by increasing laser energy absorption.
• Vacuum quality by acting as a pump.
• Salt tolerance by selectively neutralizing contaminants.

Correct Answer: Mass resolution and mass accuracy by correcting for kinetic energy spread of ions.

Q20. What is the principal challenge for quantitative analysis using MALDI in pharmaceutical studies?

• Shot-to-shot and spot-to-spot variability from heterogeneous matrix crystallization and ion suppression, which complicates reproducible quantitation.
• Inability to detect peptides and proteins at nanomolar concentrations.
• Complete incompatibility with internal standards.
• Requirement to analyze only under ambient pressure conditions.

Correct Answer: Shot-to-shot and spot-to-spot variability from heterogeneous matrix crystallization and ion suppression, which complicates reproducible quantitation.

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