LC-MS/MS: instrumentation and applications MCQs With Answer

LC-MS/MS: instrumentation and applications MCQs With Answer

Liquid chromatography–tandem mass spectrometry (LC-MS/MS) is a cornerstone technique in pharmaceutical analysis, combining high-performance liquid chromatography with sensitive mass spectrometric detection. B. Pharm students should understand LC components (columns, mobile phases, gradient elution), ionization sources (ESI, APCI), mass analyzers (triple quadrupole, QTOF), and tandem MS modes (MRM/SRM, product ion scans). Key applications include bioanalysis, pharmacokinetics, therapeutic drug monitoring, metabolite identification, and impurity profiling. Practical topics—sample preparation (SPE, protein precipitation), matrix effects, internal standards, method validation (LOD, LOQ, accuracy, precision), and data processing—are essential for reliable quantitation and interpretation. Now let’s test your knowledge with 30 MCQs on this topic.

Q1. Which ionization source is most commonly used in LC-MS/MS for polar, thermally labile drug molecules?

• Electron ionization (EI)
• Electrospray ionization (ESI)
• Matrix-assisted laser desorption/ionization (MALDI)
• Fast atom bombardment (FAB)

Correct Answer: Electrospray ionization (ESI)

Q2. In a triple quadrupole (QqQ) mass spectrometer operating in MRM mode, what is the primary role of Q2?

• To separate ions by m/z (first mass filter)
• To fragment selected precursor ions (collision cell)
• To detect ions using electron multiplier
• To perform high-resolution mass measurement

Correct Answer: To fragment selected precursor ions (collision cell)

Q3. Which mobile phase additive is commonly used to enhance protonation in positive ESI for LC-MS/MS bioanalysis?

• Trifluoroacetic acid (TFA)
• Formic acid
• Sodium hydroxide
• Dimethyl sulfoxide (DMSO)

Correct Answer: Formic acid

Q4. What does MRM stand for and why is it preferred for quantitative assays?

• Multiple Reaction Monitoring; high selectivity and sensitivity for targeted quantitation
• Mass Range Measurement; broad detection of unknowns
• Matrix Reaction Method; reduces matrix effects
• Maximum Response Mode; increases instrument speed

Correct Answer: Multiple Reaction Monitoring; high selectivity and sensitivity for targeted quantitation

Q5. Which sample preparation technique provides the best cleanup and concentration for trace drug analysis in plasma?

• Protein precipitation (PPT)
• Solid-phase extraction (SPE)
• Dilute-and-shoot
• Direct injection without pretreatment

Correct Answer: Solid-phase extraction (SPE)

Q6. What is ion suppression in LC-MS/MS and a common cause?

• Loss of chromatographic resolution due to column aging
• Decrease in ionization efficiency caused by co-eluting matrix components
• Electrical failure in the detector
• Incorrect software peak integration

Correct Answer: Decrease in ionization efficiency caused by co-eluting matrix components

Q7. Which stationary phase is most widely used for reversed-phase LC in drug analysis?

• Strong cation exchange (SCX)
• Hydrophilic interaction (HILIC)
• C18 (octadecylsilane)
• Size-exclusion packing

Correct Answer: C18 (octadecylsilane)

Q8. For quantitation, why are stable isotope-labeled internal standards preferred?

• They increase chromatographic retention time
• They correct for matrix effects and losses during sample preparation
• They eliminate the need for calibration curves
• They reduce instrument maintenance frequency

Correct Answer: They correct for matrix effects and losses during sample preparation

Q9. What parameter defines the lowest concentration that can be quantified with acceptable accuracy and precision?

• Limit of detection (LOD)
• Limit of quantification (LOQ or LLOQ)
• Upper limit of linearity (ULOQ)
• Signal-to-noise ratio (S/N) only

Correct Answer: Limit of quantification (LOQ or LLOQ)

Q10. In collision-induced dissociation (CID), what is adjusted to optimize fragmentation?

• Column temperature
• Collision energy (CE)
• Mobile phase pH
• Spray voltage only

Correct Answer: Collision energy (CE)

Q11. Which LC gradient strategy helps separate early-eluting polar metabolites from late-eluting nonpolar drugs?

• Isocratic elution with high organic content
• Step gradient without equilibration
• Shallow linear gradient from aqueous to organic
• Using only buffer without organic modifier

Correct Answer: Shallow linear gradient from aqueous to organic

Q12. What is the major advantage of high-resolution mass spectrometry (HRMS) like QTOF or Orbitrap in drug metabolism studies?

• Lower instrument cost compared to triple quadrupole
• Ability to obtain accurate mass for elemental composition and unknown identification
• Simpler user interface for routine MRM assays
• Less susceptibility to matrix effects

Correct Answer: Ability to obtain accurate mass for elemental composition and unknown identification

Q13. Which parameter should be minimized to allow more MRM transitions per cycle without losing sensitivity?

• Dwell time per transition
• Column length
• Mobile phase ionic strength
• Flow cell volume

Correct Answer: Dwell time per transition

Q14. During method validation, which experiment assesses whether the method can selectively quantify analyte in presence of endogenous compounds?

• Precision and accuracy test
• Matrix selectivity/specificity study
• System suitability only
• Carryover test alone

Correct Answer: Matrix selectivity/specificity study

Q15. Which mobile phase salt is commonly used for negative ion mode ESI to improve deprotonation?

• Ammonium acetate
• Potassium chloride
• Calcium carbonate
• Nitric acid

Correct Answer: Ammonium acetate

Q16. What is scheduled MRM and its main benefit in quantitative LC-MS/MS?

• Monitoring MRMs continuously across the chromatogram to maximize sensitivity
• Acquiring MRMs only around expected retention times to increase cycle dwell time and transitions
• Running full-scan MS to discover unknowns
• Using a single universal MRM for all analytes

Correct Answer: Acquiring MRMs only around expected retention times to increase cycle dwell time and transitions

Q17. In LC-MS/MS, what is the common effect of using trifluoroacetic acid (TFA) in mobile phase for ESI?

• Improves ESI signal sensitivity for all compounds
• Causes ion suppression and reduced MS sensitivity
• Increases volatility and enhances ionization
• Eliminates need for internal standards

Correct Answer: Causes ion suppression and reduced MS sensitivity

Q18. Which parameter is most critical to monitor for carryover risk in LC-MS/MS assays?

• Column particle size only
• Blank injection after high-concentration samples
• Spray chamber temperature only
• Detector voltage daily

Correct Answer: Blank injection after high-concentration samples

Q19. For polar basic drugs with poor retention on C18, which chromatographic mode is useful?

• Reverse-phase with higher aqueous content only
• Hydrophilic interaction liquid chromatography (HILIC)
• Size-exclusion chromatography
• Supercritical fluid chromatography exclusively

Correct Answer: Hydrophilic interaction liquid chromatography (HILIC)

Q20. What is the main purpose of using multiple reaction monitoring transitions (quantifier and qualifier) in a quantitative method?

• To reduce analysis time by half
• To confirm analyte identity and improve specificity
• To avoid using internal standards
• To increase ion suppression intentionally

Correct Answer: To confirm analyte identity and improve specificity

Q21. Which factor most improves method sensitivity in LC-MS/MS when analyzing low-abundance drugs?

• Increasing injection volume without cleanup
• Efficient sample cleanup (SPE) and use of stable isotope IS
• Using non-volatile buffer salts
• Reducing column temperature to 0°C

Correct Answer: Efficient sample cleanup (SPE) and use of stable isotope IS

Q22. When performing pharmacokinetic (PK) studies, which LC-MS/MS attribute is most critical?

• High-resolution images of chromatograms
• Accurate, precise quantitation across the expected concentration range
• Ability to run isocratic methods only
• Exclusively qualitative metabolite profiling

Correct Answer: Accurate, precise quantitation across the expected concentration range

Q23. In LC-MS/MS method validation, which test evaluates reproducibility over different days and operators?

• Intra-day precision
• Inter-day (intermediate) precision
• System suitability only
• Calibration curve linearity only

Correct Answer: Inter-day (intermediate) precision

Q24. Which scan type is most appropriate for screening unknown metabolites in an untargeted workflow?

• Targeted MRM with fixed transitions
• Full-scan high-resolution MS with data-dependent MS/MS (DDA)
• Selected ion monitoring (SIM)
• Single ion reaction monitoring

Correct Answer: Full-scan high-resolution MS with data-dependent MS/MS (DDA)

Q25. What is the effect of increasing sheath gas or nebulizer gas in an ESI source?

• No impact on spray desolvation
• Improves desolvation and can increase signal, up to a point
• Always decreases ionization efficiency
• Makes the mass analyzer non-functional

Correct Answer: Improves desolvation and can increase signal, up to a point

Q26. Which parameter describes the mass resolving power necessary to distinguish two ions of similar m/z?

• Signal-to-noise ratio
• Resolution (R)
• Chromatographic retention factor (k’)
• Plate count (N)

Correct Answer: Resolution (R)

Q27. Which approach helps reduce matrix effects during LC-MS/MS bioanalysis?

• Using non-volatile salts to improve retention
• Optimized sample cleanup, chromatographic separation, and stable isotope IS
• Increasing sample injection volume indefinitely
• Always using TFA in mobile phase

Correct Answer: Optimized sample cleanup, chromatographic separation, and stable isotope IS

Q28. In method transfer between labs, which parameter must be matched to reproduce retention times and MS response?

• Column dimensions and stationary phase chemistry, mobile phase composition, and flow rate
• Only instrument brand matters
• Laboratory room humidity exclusively
• Software version only

Correct Answer: Column dimensions and stationary phase chemistry, mobile phase composition, and flow rate

Q29. What does the term “selectivity” in LC-MS/MS method validation refer to?

• Ability to measure analyte without interference from other components
• Maximum possible signal intensity
• Speed at which samples are injected
• Cost-effectiveness of consumables

Correct Answer: Ability to measure analyte without interference from other components

Q30. Which application of LC-MS/MS is essential in forensic toxicology?

• Quantitative determination of recreational drugs and metabolites in biological matrices
• Measuring UV absorbance of tablets only
• Assessing tablet hardness in manufacturing
• Counting colony forming units in microbiology

Correct Answer: Quantitative determination of recreational drugs and metabolites in biological matrices

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