Metabolite identification – In-vitro approaches MCQs With Answer

Introduction: Metabolite identification – In-vitro approaches MCQs With Answer is designed for M.Pharm students to strengthen understanding of modern strategies used to characterize drug metabolism in vitro. This short quiz collection covers practical and theoretical aspects: common in vitro systems (microsomes, hepatocytes, S9, recombinant enzymes), phase I/II enzyme cofactors, trapping of reactive intermediates, sample preparation, and analytical workflows using LC–MS/MS and high‑resolution MS. Questions emphasize interpretation of metabolic pathways, reaction phenotyping, IVIVE concepts, and pitfalls such as non‑specific binding and artifact formation. Use these MCQs to test critical thinking and apply bioanalytical principles in metabolite identification and drug safety evaluation.

Q1. Which in‑vitro system best retains both phase I and phase II metabolic activities and cellular transporters similar to in vivo liver?

• Human liver microsomes
• Recombinant CYP enzymes
• Cryopreserved human hepatocytes
• Rat S9 fraction

Correct Answer: Cryopreserved human hepatocytes

Q2. What cofactor is essential for UDP‑glucuronosyltransferase (UGT) mediated glucuronidation in in‑vitro assays?

• NADPH
• UDP‑glucuronic acid (UDPGA)
• PAPS
• GSH

Correct Answer: UDP‑glucuronic acid (UDPGA)

Q3. Which trapping agent is commonly used to detect electrophilic reactive metabolites by forming stable conjugates?

• Isocyanate
• Glutathione (GSH)
• Acetic anhydride
• Formaldehyde

Correct Answer: Glutathione (GSH)

Q4. For reaction phenotyping of cytochrome P450 (CYP) enzymes, which approach best identifies the specific human CYP isoforms responsible for metabolism?

• Use of pooled human liver microsomes only
• Chemical inhibition profiling with selective CYP inhibitors and recombinant CYP enzymes
• Measuring plasma clearance in animals
• Using rat hepatocytes without human cofactors

Correct Answer: Chemical inhibition profiling with selective CYP inhibitors and recombinant CYP enzymes

Q5. Which analytical technique provides the highest confidence for elemental composition and accurate mass determination of unknown metabolites?

• Triple quadrupole LC–MS in SRM mode
• Gas chromatography with FID
• High‑resolution mass spectrometry (HRMS) such as Orbitrap or TOF
• UV spectroscopy

Correct Answer: High‑resolution mass spectrometry (HRMS) such as Orbitrap or TOF

Q6. In microsomal incubations, the presence of the NADPH‑regenerating system primarily supports which metabolic reactions?

• UDP‑glucuronidation
• Sulfation
• CYP‑mediated oxidative (phase I) reactions
• Glutathione conjugation

Correct Answer: CYP‑mediated oxidative (phase I) reactions

Q7. Which parameter is most directly estimated from in‑vitro intrinsic clearance (Clint) to predict hepatic clearance by IVIVE?

• Maximum tolerated dose
• Fraction unbound in plasma only
• Hepatic intrinsic clearance after scaling with microsomal protein per gram liver and liver weight
• Volume of distribution

Correct Answer: Hepatic intrinsic clearance after scaling with microsomal protein per gram liver and liver weight

Q8. What is the purpose of using heat‑inactivated control incubations in metabolite identification studies?

• To enhance enzyme activity
• To quantify non‑enzymatic degradation and artefact formation
• To provide an internal standard for MS analysis
• To increase protein binding

Correct Answer: To quantify non‑enzymatic degradation and artefact formation

Q9. When performing LC‑MS metabolite profiling, which data acquisition mode improves the chance of detecting unexpected metabolites without predefined transitions?

• Targeted SRM/MRM
• Data‑dependent acquisition (DDA) or full‑scan HRMS
• UV diode array only
• Fluorescence detection

Correct Answer: Data‑dependent acquisition (DDA) or full‑scan HRMS

Q10. Which approach helps distinguish between metabolite peaks and in‑source fragment ions during MS analysis?

• Increasing source temperature indefinitely
• Performing MS/MS (product ion spectra) and evaluating chromatographic retention times
• Only monitoring nominal mass
• Using no chromatographic separation

Correct Answer: Performing MS/MS (product ion spectra) and evaluating chromatographic retention times

Q11. Which cofactor is required for sulfation reactions catalyzed by sulfotransferases in in‑vitro incubations?

• UDPGA
• NADH
• PAPS (3′‑phosphoadenosine‑5′‑phosphosulfate)
• ATP

Correct Answer: PAPS (3′‑phosphoadenosine‑5′‑phosphosulfate)

Q12. In metabolite identification, mass defect filtering is used primarily to:

• Improve chromatographic resolution
• Enrich for signals corresponding to metabolites with similar elemental composition motifs
• Calibrate the mass spectrometer
• Quantify parent drug concentration

Correct Answer: Enrich for signals corresponding to metabolites with similar elemental composition motifs

Q13. Which of the following experimental considerations reduces false negatives due to non‑specific binding of compound to incubation vessels or microsomes?

• Using extremely low protein concentrations without adjustment
• Adding albumin or using appropriate solvent controls and measuring unbound fraction
• Omitting cofactors to reduce complexity
• Increasing incubation pH to extreme values

Correct Answer: Adding albumin or using appropriate solvent controls and measuring unbound fraction

Q14. Which recombinant enzyme system is most suitable for confirming whether a single CYP isoform catalyzes a metabolite formation reaction?

• Pooled human liver microsomes only
• Human recombinant CYP expressed in baculovirus‑infected insect cells
• Rat S9 fraction
• Human plasma enzymes

Correct Answer: Human recombinant CYP expressed in baculovirus‑infected insect cells

Q15. A metabolite shows a +16 Da mass shift relative to parent and similar retention behavior; the most likely biotransformation is:

• Glucuronidation
• Oxidation (addition of oxygen, e.g., hydroxylation)
• Dealkylation
• Sulfation

Correct Answer: Oxidation (addition of oxygen, e.g., hydroxylation)

Q16. Which technique is particularly helpful to localize the site of modification on a metabolite when multiple possible positions exist?

• Integration of full‑scan MS only
• MSn (tandem MSn) fragmentation mapping and comparison with synthetic standards
• UV‑vis absorption maximum
• Simple retention time comparison without MS/MS

Correct Answer: MSn (tandem MSn) fragmentation mapping and comparison with synthetic standards

Q17. In vitro covalent binding studies frequently use radioisotopes. What is the primary concern assessed by these studies?

• Metabolic half‑life in plasma
• Extent of irreversible binding of reactive metabolites to proteins and potential toxicity risk
• Solubility of the parent drug
• Thermal stability of enzymes

Correct Answer: Extent of irreversible binding of reactive metabolites to proteins and potential toxicity risk

Q18. When scaling in‑vitro clearance to predict human hepatic clearance, which additional parameter is crucial for converting intrinsic clearance to hepatic clearance using the well‑stirred model?

• Fraction metabolized by kidneys
• Fraction unbound in plasma (fu) and hepatic blood flow
• Lipophilicity (logP) only
• pKa of the parent compound

Correct Answer: Fraction unbound in plasma (fu) and hepatic blood flow

Q19. Which artifact can arise from using methanol or acetonitrile during sample quench that may complicate metabolite identification?

• Formation of solvent adducts or reactive artifacts such as methylation/acetylation
• Improved enzymatic activity
• Complete elimination of all metabolites
• Automatic correction of mass calibration

Correct Answer: Formation of solvent adducts or reactive artifacts such as methylation/acetylation

Q20. For confident structural assignment of a phase II metabolite (e.g., glucuronide), which complementary approach increases certainty?

• Reliance solely on nominal mass increase
• Enzymatic hydrolysis with β‑glucuronidase combined with MS and comparison to aglycone
• Only measuring in plasma without MS/MS
• Ignoring retention time shifts

Correct Answer: Enzymatic hydrolysis with β‑glucuronidase combined with MS and comparison to aglycone

Download