Metabolite identification – In-vivo approaches MCQs With Answer

Introduction: Metabolite identification – In-vivo approaches MCQs With Answer is designed for M.Pharm students preparing for advanced courses and competitive exams in Modern Bio-Analytical Techniques (MPA 202T). This set covers principles and practical aspects of in-vivo metabolite identification: sampling strategies (plasma, urine, bile, tissues), radiolabeling and stable isotope studies, mass balance and microdialysis, sample preparation methods, and modern mass spectrometric tools (HRMS, MSn, LC-MS/MS). Questions emphasize regulatory concepts such as MIST, species selection, and safety-related metabolite testing, plus data interpretation for structural elucidation of metabolites. These MCQs foster critical thinking needed for designing, executing, and interpreting in-vivo metabolite studies.

Q1. Which in-vivo sampling method provides continuous localized extracellular fluid sampling for pharmacokinetic and metabolite studies?

• Portal vein cannulation
• Bile duct cannulation
• Microdialysis
• Plasma centrifugation

Correct Answer: Microdialysis

Q2. In mass balance studies using radiolabeled drug, the primary objective is to:

• Quantify only parent drug in plasma
• Determine total recovery of radioactivity in excreta to characterize disposition
• Identify protein binding in vitro
• Assess tissue accumulation by histology

Correct Answer: Determine total recovery of radioactivity in excreta to characterize disposition

Q3. Which type of radiolabel is most commonly used for quantitative in-vivo metabolite tracking due to its stability in carbon backbone?

• 35S
• 3H (tritium)
• 14C
• 32P

Correct Answer: 14C

Q4. The “Metabolites in Safety Testing” (MIST) guidance primarily addresses:

• Analytical method validation for LC-MS assays
• Identification of metabolites formed at higher exposure in humans than in animals and their safety assessment
• Good Laboratory Practices for radiolabel handling
• Standard operating procedures for tissue homogenization

Correct Answer: Identification of metabolites formed at higher exposure in humans than in animals and their safety assessment

Q5. Which MS feature is most critical for determining elemental composition of an unknown in-vivo metabolite?

• Triple quadrupole selected reaction monitoring
• High-resolution accurate mass measurement
• Ion trap low-resolution spectra
• UV absorbance spectrum

Correct Answer: High-resolution accurate mass measurement

Q6. Which sample preparation technique is preferred to remove proteins while retaining small molecule metabolites prior to LC-MS analysis?

• Solid-phase extraction (SPE)
• Protein precipitation with organic solvent
• Ultracentrifugation at 100,000 x g
• Lyophilization without reconstitution

Correct Answer: Protein precipitation with organic solvent

Q7. Bile duct cannulation in rodent studies is most useful to:

• Collect cerebrospinal fluid for CNS metabolites
• Directly measure hepatic excretion and enterohepatic metabolites
• Monitor renal clearance with high sensitivity
• Obtain arterial blood for parent drug PK

Correct Answer: Directly measure hepatic excretion and enterohepatic metabolites

Q8. When using stable isotope labeling (e.g., 13C, 15N) for metabolite identification, a major advantage is:

• Radioactive decay improves signal-to-noise ratio
• Isotopic shifts allow clear differentiation of drug-derived metabolites from endogenous background
• It eliminates need for chromatographic separation
• It increases ionization efficiency uniformly for all metabolites

Correct Answer: Isotopic shifts allow clear differentiation of drug-derived metabolites from endogenous background

Q9. Which analytical approach is best for structural elucidation of phase II conjugates formed in vivo?

• GC-MS with electron impact after derivatization
• LC-HRMS with MS/MS and diagnostic neutral loss scanning
• Fluorescence detection
• Enzyme-linked immunosorbent assay (ELISA)

Correct Answer: LC-HRMS with MS/MS and diagnostic neutral loss scanning

Q10. In vivo portal vein sampling in preclinical studies provides special insight into:

• Renal clearance mechanisms
• Hepatic first-pass metabolism and pre-systemic metabolite formation
• Cerebral distribution of metabolites
• Biliary excretion rate constants

Correct Answer: Hepatic first-pass metabolism and pre-systemic metabolite formation

Q11. A common challenge in identifying low-abundance in-vivo metabolites is:

• Too high signal due to radiolabel incorporation
• Matrix interferences and ion suppression masking metabolite signals
• Excessive chromatographic resolution making peaks indistinguishable
• Lack of any endogenous metabolites in biological matrices

Correct Answer: Matrix interferences and ion suppression masking metabolite signals

Q12. Which technique allows spatial mapping of drug-related radioactivity in tissues after in-vivo dosing?

• Microdialysis sampling
• Autoradiography
• Protein precipitation
• Direct infusion ESI-MS without separation

Correct Answer: Autoradiography

Q13. For confident in-vivo metabolite identification, which combination of evidence is typically required?

• Retention time only
• Accurate mass, MS/MS fragmentation pattern, and chromatographic retention behavior
• Only UV absorbance and colorimetric response
• Elution volume from gel filtration exclusively

Correct Answer: Accurate mass, MS/MS fragmentation pattern, and chromatographic retention behavior

Q14. Which metabolic pathway would likely produce a metabolite with a mass increase of +176 Da?

• Hydroxylation (phase I)
• Glucuronidation (addition of glucuronic acid)
• Methylation
• Dealkylation

Correct Answer: Glucuronidation (addition of glucuronic acid)

Q15. In vivo studies indicate a human metabolite constitutes >10% of total circulating drug-related material but is absent in preclinical species; regulatory significance per MIST is that:

• No further action is required
• Additional nonclinical safety testing of the human-specific metabolite may be required
• The metabolite should be ignored if parent drug is active
• Only in vitro testing in bacteria is necessary

Correct Answer: Additional nonclinical safety testing of the human-specific metabolite may be required

Q16. When interpreting MS/MS spectra of metabolites, neutral loss of 80 Da commonly suggests which conjugation?

• Sulfation (loss of SO3/HSO4 equivalent)
• Glucuronidation
• Acetylation
• Hydrogenation

Correct Answer: Sulfation (loss of SO3/HSO4 equivalent)

Q17. Which approach improves detection of polar phase II metabolites in LC-MS analysis?

• Using reversed-phase C18 only without modifiers
• Applying hydrophilic interaction chromatography (HILIC) or polar-embedded phases and appropriate mobile phases
• Direct infusion without chromatography
• Using GC-MS without derivatization

Correct Answer: Applying hydrophilic interaction chromatography (HILIC) or polar-embedded phases and appropriate mobile phases

Q18. A radiolabeled mass balance study reports 85% of dose recovered in feces and 10% in urine. This suggests the primary elimination route is:

• Renal excretion
• Biliary and fecal excretion
• Lung exhalation
• Metabolic trapping in plasma proteins

Correct Answer: Biliary and fecal excretion

Q19. In structural confirmation of a suspected metabolite, synthesizing a reference standard is valuable because it allows:

• Decreasing instrument sensitivity
• Comparison of chromatographic retention time and MS/MS spectra to confirm identity
• Elimination of need for regulatory reporting
• Replacing in-vivo studies entirely

Correct Answer: Comparison of chromatographic retention time and MS/MS spectra to confirm identity

Q20. Which in-vivo experimental design element helps distinguish between metabolites formed by host enzymes versus gut microbiota?

• Comparing plasma samples only at terminal timepoint
• Conducting studies in germ-free or antibiotic-treated animals versus conventional animals
• Using only in vitro hepatocyte studies
• Measuring only total radioactivity without metabolite profiling

Correct Answer: Conducting studies in germ-free or antibiotic-treated animals versus conventional animals

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