LC-MS in bioactivity screening MCQs With Answer

Introduction: LC-MS (liquid chromatography–mass spectrometry) has become an indispensable tool in bioactivity screening for M. Pharm students, enabling sensitive detection, identification and quantitation of bioactive molecules from complex biological matrices. This blog provides focused MCQs to reinforce understanding of LC-MS principles, ionization techniques, mass analyzers, tandem MS strategies, sample preparation, data interpretation and common pitfalls such as ion suppression and matrix effects. Questions emphasize practical aspects used in bioassay-guided fractionation, dereplication, targeted quantitation (MRM/SRM) and high-resolution mass spectrometry for structure elucidation. These MCQs are designed to prepare students for examinations and real-world screening workflows in drug discovery and pharmacognosy.

Q1. What is the primary advantage of coupling liquid chromatography (LC) with mass spectrometry (MS) in bioactivity screening?

• Faster run times than either technique alone
• Ability to separate complex mixtures and provide molecular mass and structural information
• Lower cost per analysis compared to UV detection
• Eliminates the need for sample preparation

Correct Answer: Ability to separate complex mixtures and provide molecular mass and structural information

Q2. Which ionization technique is most commonly used for polar, thermally labile bioactive compounds in LC-MS?

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

Correct Answer: Electrospray ionization (ESI)

Q3. In targeted quantitative bioactivity screening where high sensitivity and selectivity are required, which MS acquisition mode is preferred?

• Full-scan high-resolution MS
• Selected ion monitoring (SIM)
• Multiple reaction monitoring (MRM/SRM) on a triple quadrupole
• Neutral loss scan

Correct Answer: Multiple reaction monitoring (MRM/SRM) on a triple quadrupole

Q4. Which mass analyzer provides the highest mass accuracy and resolving power commonly used for dereplication and elemental composition determination?

• Quadrupole
• Ion trap
• Time-of-flight (TOF) or Orbitrap (high-resolution MS)
• Magnetic sector low-resolution

Correct Answer: Time-of-flight (TOF) or Orbitrap (high-resolution MS)

Q5. What is ‘dereplication’ in the context of LC-MS bioactivity screening?

• The process of increasing sample concentration for better detection
• The identification of previously known compounds early to avoid re-isolation
• Removing salts from samples before LC-MS
• Automated fragmentation of unknowns for structure elucidation

Correct Answer: The identification of previously known compounds early to avoid re-isolation

Q6. Which factor most commonly causes ion suppression in LC-ESI-MS when analyzing biological extracts?

• High-resolution mass analyzer
• Co-eluting matrix components such as salts, lipids or detergents
• Using a narrow-bore LC column
• Low sample temperature

Correct Answer: Co-eluting matrix components such as salts, lipids or detergents

Q7. In tandem mass spectrometry (MS/MS) for structural elucidation, what does collision-induced dissociation (CID) achieve?

• Concentrates ions before detection
• Causes precursor ions to fragment into product ions for structural information
• Separates ions by mobility in a gas
• Converts neutral molecules to ions

Correct Answer: Causes precursor ions to fragment into product ions for structural information

Q8. When performing LC-MS-based metabolite profiling for bioactivity, why is high-resolution accurate mass (HRAM) important?

• It reduces run time dramatically
• It allows precise determination of elemental compositions and distinguishes isobaric species
• It eliminates the need for chromatographic separation
• It increases ion suppression

Correct Answer: It allows precise determination of elemental compositions and distinguishes isobaric species

Q9. Which sample preparation technique is commonly used to remove proteins before LC-MS analysis of biological extracts?

• Solid-phase microextraction (SPME)
• Protein precipitation using organic solvents (e.g., acetonitrile)
• Lyophilization without cleanup
• Direct infusion without cleanup

Correct Answer: Protein precipitation using organic solvents (e.g., acetonitrile)

Q10. For quantitation of a pharmaceutical compound in plasma using LC-MS, what internal standard is most appropriate?

• A structurally unrelated compound not present in the sample
• A stable isotope-labeled analogue of the target compound
• Any compound with similar retention time
• An external calibration standard run separately

Correct Answer: A stable isotope-labeled analogue of the target compound

Q11. What is the main advantage of using UHPLC (ultra-high-performance LC) coupled to MS in screening workflows?

• Lower sensitivity than conventional HPLC
• Improved chromatographic resolution and faster separations, increasing throughput
• Compatibility only with low-resolution MS
• Eliminates need for ionization

Correct Answer: Improved chromatographic resolution and faster separations, increasing throughput

Q12. In LC-MS data analysis for bioactivity screening, what does extracted ion chromatogram (XIC/EIC) display?

• Complete mass spectrum averaged over the run
• Intensity vs retention time for a selected m/z value or small m/z window
• Fragmentation pattern of all ions
• The chromatogram of UV absorbance over time

Correct Answer: Intensity vs retention time for a selected m/z value or small m/z window

Q13. Which adduct formation is commonly observed in positive ESI for small organic molecules?

• M−H (deprotonated molecule)
• [M+H]+ (protonated molecule)
• [M−Na]−
• Neutral radical M•

Correct Answer: [M+H]+ (protonated molecule)

Q14. For LC-MS method validation in bioactivity screening, which parameter assesses reproducibility over different days?

• Limit of detection (LOD)
• System suitability test
• Inter-day precision (or intermediate precision)
• Mass resolution

Correct Answer: Inter-day precision (or intermediate precision)

Q15. Which approach best helps to identify unknown bioactive constituents using LC-MS and bioassay data together?

• Random fractionation without MS guidance
• Bioassay-guided fractionation combined with LC-MS based dereplication
• Only high-throughput UV screening
• Direct NMR without prior fractionation

Correct Answer: Bioassay-guided fractionation combined with LC-MS based dereplication

Q16. In LC-MS, what does the term “mass accuracy” refer to?

• The width of chromatographic peaks
• The closeness of the measured m/z value to the true theoretical m/z expressed in ppm
• The number of ions detected per second
• The resolution at which peaks can be separated in time

Correct Answer: The closeness of the measured m/z value to the true theoretical m/z expressed in ppm

Q17. Which instrument configuration is most suitable for rapid screening of known biomarkers with very low concentrations in plasma?

• Single quadrupole in full-scan mode
• Triple quadrupole operating in MRM mode
• Ion trap in MSn mode for unknowns
• GC-MS with electron ionization

Correct Answer: Triple quadrupole operating in MRM mode

Q18. When interpreting MS/MS spectra for structural clues of a bioactive natural product, which fragment type is most informative?

• Random noise peaks below the baseline
• Characteristic neutral losses and diagnostic product ions
• Only the molecular ion without fragments
• Adduct peaks unrelated to structure

Correct Answer: Characteristic neutral losses and diagnostic product ions

Q19. Which database/resource is commonly used for matching LC-HRMS accurate masses of natural products for dereplication?

• PubChem and specialized natural product databases like DNP or GNPS libraries
• Only general search engines like Google
• Textbooks without spectral data
• Clinical trial registries

Correct Answer: PubChem and specialized natural product databases like DNP or GNPS libraries

Q20. In bioactivity screening workflows using LC-MS, why is orthogonal separation (e.g., reversed-phase followed by HILIC) sometimes applied?

• To increase ion suppression deliberately
• To improve the separation of chemically diverse compounds and reduce co-elution, enhancing detection
• To reduce analysis time by combining two similar separations
• To avoid tandem MS experiments

Correct Answer: To improve the separation of chemically diverse compounds and reduce co-elution, enhancing detection

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