Protocols for metabolite identification MCQs With Answer

Introduction: Protocols for metabolite identification MCQs With Answer is designed to help M.Pharm students build a practical and conceptual foundation in metabolite identification techniques used in modern drug development. This blog presents focused multiple-choice questions that cover experimental design, in vitro and in vivo models, sample preparation methods, chromatographic and mass spectrometric strategies, data interpretation, trapping of reactive metabolites, and regulatory considerations. Each question emphasizes real-world protocols, analytical challenges, and decision-making steps in metabolite profiling. By practicing these MCQs, students will reinforce core principles, recognize common pitfalls, and prepare for laboratory work and examinations in modern bio-analytical techniques.

Q1. Which in vitro system is most commonly used for initial human metabolic clearance screening because it contains a broad range of drug-metabolizing enzymes including CYPs and UGTs?

• Recombinant single CYP isozyme preparation
• Human liver microsomes
• Human plasma
• Reconstituted UDP-glucuronosyltransferase complex alone

Correct Answer: Human liver microsomes

Q2. What is the main advantage of using cryopreserved human hepatocytes over liver microsomes for metabolite identification?

• Higher throughput due to easier handling
• Contain both phase I and phase II enzymes and transporters, allowing more complete metabolic profiling
• Provide only oxidative metabolism simplifying data analysis
• Require no co-factors for conjugation reactions

Correct Answer: Contain both phase I and phase II enzymes and transporters, allowing more complete metabolic profiling

Q3. In LC-HRMS-based metabolite identification, why is high-resolution accurate mass (HRAM) important?

• It eliminates the need for chromatography
• It provides accurate elemental composition and mass defect information that aids structural proposals
• It reduces sample preparation time
• It limits data to parent ion only, avoiding complex spectra

Correct Answer: It provides accurate elemental composition and mass defect information that aids structural proposals

Q4. Which trapping agent is commonly used to detect electrophilic reactive metabolites such as iminium or quinone species in in vitro assays?

• Glutathione (GSH)
• Sodium chloride
• Bovine serum albumin
• EDTA

Correct Answer: Glutathione (GSH)

Q5. During sample preparation for metabolite analysis by LC-MS, protein precipitation is often followed by which step to improve sensitivity and reduce matrix effects?

• Direct injection without concentration
• Solid-phase extraction or SPE cleanup
• Dilution with water only
• Freeze-thaw cycles

Correct Answer: Solid-phase extraction or SPE cleanup

Q6. Which experimental approach most directly identifies which specific CYP isoforms metabolize a drug?

• Metabolic stability in pooled human plasma
• Incubations with recombinant human CYP isoforms or chemical inhibition studies
• Whole blood stability assay at 37°C
• Passive diffusion assay across artificial membranes

Correct Answer: Incubations with recombinant human CYP isoforms or chemical inhibition studies

Q7. What is the primary reason to include stable-isotope labeled analogs (e.g., 13C, 15N) of the parent drug in metabolite identification studies?

• To increase chromatographic retention time
• To facilitate discrimination of drug-derived signals from background endogenous peaks and confirm metabolite origin
• To inhibit metabolism
• To replace the need for MS/MS experiments

Correct Answer: To facilitate discrimination of drug-derived signals from background endogenous peaks and confirm metabolite origin

Q8. For structural elucidation of a novel metabolite, which complementary technique to LC-MS is most informative for confirming exact structural connectivity and stereochemistry?

• NMR spectroscopy
• Infrared (IR) spectroscopy alone
• UV-Vis spectrophotometry only
• Thin layer chromatography (TLC)

Correct Answer: NMR spectroscopy

Q9. In mass spectrometric fragmentation for metabolite ID, what does a neutral loss of 176 Da typically indicate in positive ion mode analysis?

• Loss of sulfate conjugate (SO3)
• Loss of glucuronic acid (glucuronide) moiety
• Loss of water molecule
• Loss of methyl group

Correct Answer: Loss of glucuronic acid (glucuronide) moiety

Q10. Which in vivo sample is most commonly used when assessing human metabolite profiles during clinical studies for metabolite identification and quantification?

• Urine and plasma samples across time points
• Hair and nail clippings only
• Cerebrospinal fluid exclusively
• Saliva only

Correct Answer: Urine and plasma samples across time points

Q11. What is the purpose of performing time-course incubations in microsomal or hepatocyte systems during metabolite identification?

• To determine chromatographic peak shape only
• To observe formation and disappearance kinetics of metabolites and support pathway elucidation
• To sterilize the sample
• To measure protein binding exclusively

Correct Answer: To observe formation and disappearance kinetics of metabolites and support pathway elucidation

Q12. Which data acquisition strategy in LC-MS is most suited for unbiased metabolite identification across unknown biotransformations?

• Targeted multiple reaction monitoring (MRM) only
• Data-dependent acquisition (DDA) or data-independent acquisition (DIA) full-scan HRMS strategies
• Single ion monitoring (SIM) for parent ion only
• UV detection without MS

Correct Answer: Data-dependent acquisition (DDA) or data-independent acquisition (DIA) full-scan HRMS strategies

Q13. When interpreting MS/MS spectra for a suspected hydroxylated metabolite, which of the following observations would most strongly support the site of hydroxylation?

• Complete loss of the parent ion only
• Shift in fragment masses by +16 Da localized to fragments containing the modified region
• Increase in retention time without any mass shift
• Absence of any fragment ions

Correct Answer: Shift in fragment masses by +16 Da localized to fragments containing the modified region

Q14. Which regulatory concept requires special attention when a drug produces human metabolites at levels significantly greater than in the animal species used for toxicology studies?

• Bioavailability equivalence
• Metabolites in safety testing (MIST) — assessment of human-specific metabolites
• Pharmacokinetic non-linearity only
• Compound solubility testing

Correct Answer: Metabolites in safety testing (MIST) — assessment of human-specific metabolites

Q15. Which control experiment helps confirm that a detected metabolite is produced enzymatically and not by non-enzymatic degradation in an in vitro assay?

• Including heat-inactivated microsomes or hepatocytes as negative control
• Omitting chromatographic separation
• Using double the concentration of substrate only
• Adding more organic solvent to the incubation

Correct Answer: Including heat-inactivated microsomes or hepatocytes as negative control

Q16. What is the main drawback of using recombinant single-enzyme systems to predict human in vivo metabolism?

• They perfectly mimic in vivo transporter interactions
• They may not reproduce enzyme interactions, cofactor availability, and cellular context present in vivo leading to incomplete metabolic pathways
• They are less specific for identifying isoform contributions
• They always overestimate glucuronidation rates

Correct Answer: They may not reproduce enzyme interactions, cofactor availability, and cellular context present in vivo leading to incomplete metabolic pathways

Q17. In LC-MS analysis, matrix effects primarily affect which aspect of quantitative and qualitative metabolite assessment?

• Enhancement or suppression of ionization causing inaccurate peak intensities and biases in identification
• Only UV detection sensitivity
• Chromatographic column lifetime only
• Mass resolution but not ion intensity

Correct Answer: Enhancement or suppression of ionization causing inaccurate peak intensities and biases in identification

Q18. Which experimental modification helps detect unstable or short-lived metabolites formed during in vitro incubations?

• Long incubation times at high temperature
• Addition of trapping reagents (e.g., GSH, semicarbazide) or rapid quench and cold extraction
• Removing cofactors to prevent formation
• Using higher pH exclusively

Correct Answer: Addition of trapping reagents (e.g., GSH, semicarbazide) or rapid quench and cold extraction

Q19. What role does accurate chromatographic separation play in metabolite identification workflows using LC-HRMS?

• It is unnecessary because MS resolves all isomers
• It separates isomeric metabolites, reduces ion suppression, and improves spectral clarity for structural assignments
• It only affects solvent consumption
• It prevents any need for MS/MS experiments

Correct Answer: It separates isomeric metabolites, reduces ion suppression, and improves spectral clarity for structural assignments

Q20. When reporting metabolite identification data for regulatory submission, which element is essential to include?

• Only chromatograms without experimental details
• Comprehensive methods, raw and processed MS data, identification justification, and quantitative levels with study conditions
• Only predicted structures without supporting spectra
• Only animal species name without sample provenance

Correct Answer: Comprehensive methods, raw and processed MS data, identification justification, and quantitative levels with study conditions

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