In-vitro assay of drug metabolites MCQs With Answer

Introduction: In-vitro assay of drug metabolites MCQs With Answer provides M.Pharm students with focused practice on bioanalytical strategies used to generate, detect, quantify and interpret drug metabolites in vitro. This collection emphasizes key experimental systems (microsomes, S9, hepatocytes, recombinant enzymes), essential cofactors, trapping reagents, analytical workflows (LC–MS/MS, HRMS, NMR), and translational concepts such as in vitro–in vivo extrapolation and intrinsic clearance scaling. Questions are designed to deepen understanding of assay selection, sample preparation, metabolite identification and interpretation of data for drug metabolism and safety assessment. Use these MCQs to test conceptual knowledge and prepare for practical decisions in metabolic profiling studies.

Q1. Which in-vitro system most completely represents both phase I and phase II metabolism for human drug metabolite profiling?

• Human liver microsomes
• Human hepatocytes
• Human liver S9 fraction
• Recombinant single CYP isoforms

Correct Answer: Human hepatocytes

Q2. Which cofactor is essential for cytochrome P450 (phase I) catalytic activity in microsomal incubations?

• UDP-glucuronic acid (UDPGA)
• Nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)
• 3′-Phosphoadenosine-5′-phosphosulfate (PAPS)
• Acetyl coenzyme A (Acetyl-CoA)

Correct Answer: Nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)

Q3. Which cofactor is required for glucuronidation reactions catalyzed by UDP‑glucuronosyltransferases (UGTs)?

• S-adenosylmethionine (SAM)
• UDP‑glucuronic acid (UDPGA)
• Glutathione (GSH)
• Nicotinamide adenine dinucleotide (NAD+)

Correct Answer: UDP‑glucuronic acid (UDPGA)

Q4. The S9 fraction prepared from liver homogenate contains which of the following?

• Only cytosolic enzymes
• Only microsomal enzymes
• Both microsomal and cytosolic enzymes
• Only intact hepatocytes

Correct Answer: Both microsomal and cytosolic enzymes

Q5. In a microsomal stability assay, a compound that shows high intrinsic clearance (Clint) typically indicates which in vivo characteristic?

• Likely long systemic half-life and low hepatic extraction
• Slow metabolism and high oral bioavailability
• Rapid metabolism and likely shorter in vivo half-life
• Resistance to phase I metabolism but susceptible to phase II

Correct Answer: Rapid metabolism and likely shorter in vivo half-life

Q6. What is a primary limitation of using recombinant single cytochrome P450 isoforms for metabolite identification?

• They over-represent phase II metabolism
• They do not provide isoform-specific activity
• They lack the full complement of hepatic enzymes and enzyme–enzyme interactions
• They cannot be used with NADPH

Correct Answer: They lack the full complement of hepatic enzymes and enzyme–enzyme interactions

Q7. Which trapping reagent is commonly used to capture electrophilic reactive metabolites during in‑vitro incubations?

• β-mercaptoethanol
• Glutathione (GSH)
• Sodium borohydride
• Triethylamine

Correct Answer: Glutathione (GSH)

Q8. High-resolution mass spectrometry (HRMS) provides which key advantage for metabolite identification?

• Higher dynamic range than triple quadrupole for quantification
• Accurate mass measurements enabling elemental composition determination
• Lower sensitivity for targeted low‑level metabolites
• Elimination of chromatographic separation requirements

Correct Answer: Accurate mass measurements enabling elemental composition determination

Q9. In LC–MS/MS quantification workflows, multiple reaction monitoring (MRM) transitions are primarily used to:

• Determine accurate elemental composition of unknown metabolites
• Monitor specific precursor→product ion pairs for sensitive and selective quantitation
• Provide high mass accuracy for structural elucidation
• Fragment neutral metabolites without ionization

Correct Answer: Monitor specific precursor→product ion pairs for sensitive and selective quantitation

Q10. A major advantage of using radiolabeled drug (e.g., 14C) in metabolic studies is:

• It avoids the need for chromatographic separation
• It quantifies all radiolabeled metabolites regardless of ionization efficiency in MS
• It provides exact structural information about metabolites
• It is less regulated and cheaper than mass spectrometric approaches

Correct Answer: It quantifies all radiolabeled metabolites regardless of ionization efficiency in MS

Q11. Which factors are required when performing in vitro–in vivo extrapolation (IVIVE) of hepatic clearance from microsomal intrinsic clearance?

• Plasma protein binding only
• Microsomal protein per gram liver and liver weight scaling factors
• Urinary excretion rate and glomerular filtration rate only
• Only the in vitro assay incubation volume

Correct Answer: Microsomal protein per gram liver and liver weight scaling factors

Q12. Which cofactor is necessary for sulfotransferase-mediated sulfation reactions in in‑vitro assays?

• PAPS (3′-phosphoadenosine-5′-phosphosulfate)
• UDPGA
• NADPH
• FAD

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

Q13. To study the influence of uptake and efflux transporters on metabolism, which in‑vitro model is most appropriate?

• Human liver microsomes
• Primary human hepatocytes or cultured sandwich-cultured hepatocytes
• Recombinant single CYP enzyme
• S9 fraction

Correct Answer: Primary human hepatocytes or cultured sandwich-cultured hepatocytes

Q14. For unequivocal structural elucidation of an isolated drug metabolite, including stereochemistry, which analytical technique is most definitive?

• Triple quadrupole LC–MS/MS in MRM mode
• High-resolution mass spectrometry (HRMS) alone
• Nuclear magnetic resonance spectroscopy (NMR)
• UV-visible spectroscopy

Correct Answer: Nuclear magnetic resonance spectroscopy (NMR)

Q15. Which sample preparation technique is generally most effective at minimizing matrix effects for LC–MS analysis of metabolites?

• Protein precipitation with organic solvent only
• Direct injection of crude plasma
• Solid-phase extraction (SPE)
• Dilute-and-shoot with water

Correct Answer: Solid-phase extraction (SPE)

Q16. Which selective chemical inhibitor is commonly used to probe CYP2D6-mediated metabolism in vitro?

• Ketoconazole
• Quinidine
• Furafylline
• Diclofenac

Correct Answer: Quinidine

Q17. Time-dependent inhibition (TDI) observed only after preincubation with NADPH typically indicates which phenomenon?

• Reversible competitive inhibition
• Mechanism-based (irreversible) inhibition of a P450 enzyme
• Non-specific protein binding artifact
• Loss of assay sensitivity over time

Correct Answer: Mechanism-based (irreversible) inhibition of a P450 enzyme

Q18. The fraction unbound in plasma (fu) is an important parameter because it directly affects which of the following?

• The total concentration measured by immunoassay only
• Liver microsomal protein content
• Free drug available for hepatic uptake and metabolism
• The molecular weight of the drug

Correct Answer: Free drug available for hepatic uptake and metabolism

Q19. Non-specific binding of a compound to microsomal protein during stability assays can cause which analytical artifact if not corrected?

• Overestimation of intrinsic clearance
• Complete loss of all metabolites
• Improved ionization efficiency in MS
• Conversion of phase II metabolites back to parent

Correct Answer: Overestimation of intrinsic clearance

Q20. For targeted quantification of low‑abundance metabolites with the highest sensitivity, which mass spectrometric platform is typically preferred?

• Quadrupole time-of-flight (QTOF) in full-scan mode
• Orbitrap high-resolution full-scan
• Triple quadrupole LC–MS/MS in MRM mode
• Single quadrupole with total ion monitoring

Correct Answer: Triple quadrupole LC–MS/MS in MRM mode

Download