LC-MS hyphenation MCQs With Answer

Introduction: This LC-MS hyphenation MCQ collection is designed specifically for M.Pharm students studying Advanced Instrumental Analysis (MPA 201T). It focuses on the principles and practical aspects of coupling liquid chromatography with mass spectrometry, covering ionization techniques, interfaces, mass analyzers, fragmentation, quantitative modes, matrix and solvent effects, and troubleshooting strategies common in pharmaceutical analysis. Questions emphasize deeper understanding of hyphenation challenges—ion suppression, mobile phase selection, interface tuning, and method optimization—preparing students for instrument operation, method development, and data interpretation in drug analysis, bioanalysis, and quality control labs. Use these MCQs for revision, self-assessment, and exam preparation.

Q1. What is the primary difference in the ionization mechanism between electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI)?

• ESI produces ions via gas-phase corona discharge while APCI forms ions directly from liquid droplets
• ESI generates multiply charged ions from desolvated droplets; APCI ionizes vapor-phase molecules via reagent ions
• ESI requires higher source temperatures than APCI to achieve ionization
• ESI is only suitable for nonpolar compounds while APCI is only suitable for polar compounds

Correct Answer: ESI generates multiply charged ions from desolvated droplets; APCI ionizes vapor-phase molecules via reagent ions

Q2. Which mobile phase additive is most commonly used to improve signal intensity for basic pharmaceuticals in positive ESI mode?

• Formic acid
• Ammonium acetate
• Trifluoroacetic acid (TFA)
• Sodium chloride

Correct Answer: Formic acid

Q3. In LC-MS method development, which approach best reduces ion suppression caused by co-eluting matrix components?

• Increase chromatographic flow rate to shorten analyte retention time
• Use a steeper solvent gradient to elute all analytes faster
• Optimize sample cleanup and improve chromatographic separation to separate analyte from interfering matrix components
• Add more organic solvent to the sample injection to improve desolvation

Correct Answer: Optimize sample cleanup and improve chromatographic separation to separate analyte from interfering matrix components

Q4. Which mass analyzer provides the highest mass resolving power suitable for distinguishing isobaric drug metabolites separated by a few milliDaltons?

• Quadrupole
• Ion trap
• Time-of-flight (TOF)
• Fourier transform ion cyclotron resonance (FT-ICR)

Correct Answer: Fourier transform ion cyclotron resonance (FT-ICR)

Q5. In a triple quadrupole LC-MS operating in MRM mode, what is the role of the second quadrupole (q2)?

• To separate ions by m/z for full-scan spectra
• To act as a collision cell where selected precursor ions are fragmented (CID)
• To focus ions into the detector without fragmentation
• To generate multiply charged ions from neutral molecules

Correct Answer: To act as a collision cell where selected precursor ions are fragmented (CID)

Q6. Why are ammonium salts often preferred as mobile phase buffers in LC-ESI-MS compared with nonvolatile buffers like phosphate?

• Ammonium salts increase ion suppression more than phosphate
• Ammonium salts are volatile and do not contaminate the MS source, while phosphate is nonvolatile and causes source fouling
• Phosphate buffers improve desolvation and should always be used for ESI
• Ammonium salts suppress ionization of analytes and thus improve selectivity

Correct Answer: Ammonium salts are volatile and do not contaminate the MS source, while phosphate is nonvolatile and causes source fouling

Q7. For a peptide analysis by LC-ESI-MS, which phenomenon explains the observation of multiple charge states for a single peptide?

• Peptide aggregation in solution increases m/z diversity
• Peptides acquire multiple protons at basic sites during desolvation in ESI, resulting in different charge states
• Multiple isotopes of the peptide backbone dominate the spectrum
• APCI-like gas-phase chemistry produces radical cations with varying m/z

Correct Answer: Peptides acquire multiple protons at basic sites during desolvation in ESI, resulting in different charge states

Q8. Which of the following is an advantage of polarity switching within a single LC-MS run?

• It doubles the sensitivity for all analytes
• It allows simultaneous detection of analytes ionizing better in positive and negative modes within one chromatographic run
• It reduces the need for chromatographic separation
• It eliminates matrix effects completely

Correct Answer: It allows simultaneous detection of analytes ionizing better in positive and negative modes within one chromatographic run

Q9. In high-resolution LC-MS quantitation, what is the primary benefit of using accurate mass extracted ion chromatograms (XIC) over unit resolution selected ion monitoring?

• Accurate mass XIC reduces isobaric interference and increases selectivity, improving signal-to-noise for target analytes
• Accurate mass XIC always gives higher sensitivity by increasing ion abundance
• Accurate mass XIC eliminates the need for internal standards
• Accurate mass XIC shortens chromatographic run time substantially

Correct Answer: Accurate mass XIC reduces isobaric interference and increases selectivity, improving signal-to-noise for target analytes

Q10. Which parameter most directly controls the extent of in-source fragmentation in an ESI source?

• Column particle size
• Capillary inlet/skimmer voltage and source temperature
• Mass analyzer resolution setting
• Mobile phase pH only

Correct Answer: Capillary inlet/skimmer voltage and source temperature

Q11. When performing LC-MS analysis of a basic small-molecule drug that forms sodium adducts [M+Na]+, which strategy can reduce sodium adduct formation and improve protonated molecular ion [M+H]+ intensity?

• Add excess sodium chloride to the mobile phase
• Use glassware rinsed with deionized water and add a small amount of formic acid or ammonium acetate to favor protonation
• Increase column temperature dramatically
• Switch to APCI regardless of compound polarity

Correct Answer: Use glassware rinsed with deionized water and add a small amount of formic acid or ammonium acetate to favor protonation

Q12. In LC-MS, what does the term ‘lock mass’ refer to and why is it used in high-resolution measurements?

• A calibrant ion continuously introduced to correct mass drift and improve mass accuracy during long runs
• A mass filter that blocks ions below a certain m/z to protect the detector
• A software routine that ignores low-intensity peaks during data processing
• A reagent used in the mobile phase to suppress background noise

Correct Answer: A calibrant ion continuously introduced to correct mass drift and improve mass accuracy during long runs

Q13. Which scan mode is most appropriate for highly selective quantitative measurement of trace-level drug in plasma when using a triple quadrupole?

• Full-scan MS
• Product ion scan
• Multiple reaction monitoring (MRM)
• All-ion fragmentation

Correct Answer: Multiple reaction monitoring (MRM)

Q14. What is the expected effect on chromatographic MS sensitivity when using a flow rate of 1.0 mL/min with a standard ESI source without flow splitting?

• Sensitivity typically increases because more analyte reaches the source
• Sensitivity may decrease due to incomplete desolvation and reduced ionization efficiency at high flow
• No effect on sensitivity; only retention times change
• Sensitivity becomes independent of mobile phase composition

Correct Answer: Sensitivity may decrease due to incomplete desolvation and reduced ionization efficiency at high flow

Q15. Which of the following best explains why TFA (trifluoroacetic acid) is often avoided in LC-MS mobile phases despite its chromatographic benefits?

• TFA is too volatile and causes unstable baselines
• TFA forms strong ion-pair complexes and causes severe ion suppression in ESI, reducing MS sensitivity
• TFA reacts chemically with analytes, producing artifacts
• TFA cannot be mixed with organic solvents like acetonitrile

Correct Answer: TFA forms strong ion-pair complexes and causes severe ion suppression in ESI, reducing MS sensitivity

Q16. In a Q-TOF LC-MS system, what is the main advantage of using collision-induced dissociation (CID) in the quadrupole followed by TOF detection?

• It limits mass range and reduces sensitivity for large molecules
• It allows selection of precursor ions in Q, fragmentation in collision cell, and high-resolution accurate mass measurement of fragments in TOF for confident identification
• It eliminates the need for chromatographic separation
• It reduces acquisition speed compared to single quadrupole

Correct Answer: It allows selection of precursor ions in Q, fragmentation in collision cell, and high-resolution accurate mass measurement of fragments in TOF for confident identification

Q17. Which of the following is a valid method to minimize LC-MS carryover of a strongly retained analyte?

• Reduce injection volume but keep same wash solvents
• Use stronger needle and loop wash solvents (e.g., higher organic content and additives) and optimize column wash gradient between injections
• Lower source temperature to allow analyte to adhere to surfaces more
• Use higher concentration calibration standards to mask carryover

Correct Answer: Use stronger needle and loop wash solvents (e.g., higher organic content and additives) and optimize column wash gradient between injections

Q18. When interpreting an LC-MS spectrum for a chlorinated drug metabolite, which isotopic pattern feature would strongly indicate the presence of a single chlorine atom?

• A pair of peaks separated by 2 Da with approximately 3:1 intensity ratio (M : M+2)
• A doublet with equal intensity separated by 1 Da
• A broad unresolved peak due to chlorine nuclear spin
• No isotopic peaks because chlorine is not detectable by MS

Correct Answer: A pair of peaks separated by 2 Da with approximately 3:1 intensity ratio (M : M+2)

Q19. For quantitative LC-MS assays, why are stable isotope-labeled internal standards (SIL-IS) preferred over structural analogs?

• SIL-IS coelute and exhibit nearly identical ionization and extraction behavior as the analyte, correcting for matrix effects and losses
• SIL-IS are cheaper and more readily available than structural analogs
• SIL-IS always improve chromatographic resolution
• SIL-IS eliminate the need for calibration curves

Correct Answer: SIL-IS coelute and exhibit nearly identical ionization and extraction behavior as the analyte, correcting for matrix effects and losses

Q20. In LC-MS fragmentation for structural elucidation, what is the primary difference between CID (collision-induced dissociation) in a quadrupole collision cell and HCD (higher-energy collisional dissociation) used in some orbitrap systems?

• CID uses photon energy while HCD uses collisions with neutral gas molecules
• CID typically produces lower-energy fragmentation pathways often yielding sequence-informative fragments, whereas HCD uses higher collision energies producing complementary higher-energy fragments and broader fragment coverage, often detected with high-resolution analyzers
• CID is only used for small molecules and HCD only for peptides
• There is no practical difference; the names are interchangeable

Correct Answer: CID typically produces lower-energy fragmentation pathways often yielding sequence-informative fragments, whereas HCD uses higher collision energies producing complementary higher-energy fragments and broader fragment coverage, often detected with high-resolution analyzers

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