Sample preparation for metabolite studies MCQs With Answer

Sample preparation for metabolite studies MCQs With Answer

Sample preparation is a critical step in metabolite studies because it determines the quality, reproducibility, and interpretability of downstream bioanalytical data. This short quiz collection focuses on practical and conceptual aspects of preparing biological matrices (plasma, urine, tissues) for metabolite identification and quantification using LC-MS, GC-MS and HRMS platforms. Questions cover quenching, extraction techniques (protein precipitation, liquid–liquid, solid‑phase, microextraction), enzymatic hydrolysis of conjugates, stabilization, cleanup strategies to minimize matrix effects, use of internal standards and selection of conditions that preserve labile metabolites. The MCQs are tailored for M.Pharm students preparing for advanced bioanalytical work and method development challenges.

Q1. Which initial step is most important to immediately prevent post-collection biotransformation of metabolites in a blood sample intended for metabolite profiling?

• Freeze the whole blood immediately at −20°C
• Add an organic solvent such as methanol and vortex
• Add enzyme inhibitors and rapidly cool on ice
• Dilute the blood with buffer to reduce enzyme activity

Correct Answer: Add enzyme inhibitors and rapidly cool on ice

Q2. For LC‑MS analysis of polar metabolites, which sample cleanup approach gives the best compromise between matrix removal and recovery of small polar metabolites?

• Protein precipitation with acetonitrile followed by centrifugation
• Liquid–liquid extraction using hexane
• SPE with reversed‑phase C18 cartridges only
• Direct injection of crude plasma

Correct Answer: Protein precipitation with acetonitrile followed by centrifugation

Q3. When targeting glucuronide and sulfate conjugates, which preparation step is commonly performed to quantify total (conjugated + free) metabolites?

• Solid-phase extraction on ion-exchange sorbent
• Enzymatic hydrolysis using β‑glucuronidase/arylsulfatase
• Derivatization with BSTFA for GC analysis
• Protein precipitation with perchloric acid

Correct Answer: Enzymatic hydrolysis using β‑glucuronidase/arylsulfatase

Q4. Which microextraction technique is most suitable for volatile metabolites and headspace analysis before GC‑MS?

• Solid‑phase microextraction (SPME)
• Dispersive liquid–liquid microextraction (DLLME)
• Solid-phase extraction (SPE) on C8
• Protein precipitation with methanol

Correct Answer: Solid‑phase microextraction (SPME)

Q5. What is the primary reason to include an isotopically labeled internal standard in sample preparation for quantitative metabolite analysis?

• To increase the chromatographic retention of analytes
• To correct for losses and matrix effects during sample prep and analysis
• To hydrolyze conjugates selectively
• To precipitate proteins more efficiently

Correct Answer: To correct for losses and matrix effects during sample prep and analysis

Q6. Which condition during liquid–liquid extraction (LLE) most strongly affects partitioning of a weakly basic drug metabolite?

• Salt concentration in the aqueous phase
• pH of the aqueous phase
• Type of organic cosolvent used in LC mobile phase
• Temperature of the autosampler

Correct Answer: pH of the aqueous phase

Q7. When preparing tissue homogenates for metabolite profiling, what step reduces artifactual oxidation of labile metabolites?

• Homogenization at high speed and room temperature
• Performing homogenization under inert atmosphere (nitrogen) and on ice
• Adding concentrated NaOH during homogenization
• Using only PBS buffer without antioxidants

Correct Answer: Performing homogenization under inert atmosphere (nitrogen) and on ice

Q8. Phospholipids cause matrix effects in LC‑MS. Which sample preparation approach is specifically used to remove phospholipids?

• Protein precipitation with acetonitrile alone
• Phospholipid removal plates or hybrid SPE‑phospholipid cartridges
• Liquid–liquid extraction with ethyl acetate
• Direct dilution with water

Correct Answer: Phospholipid removal plates or hybrid SPE‑phospholipid cartridges

Q9. Which statement best describes matrix‑matched calibration in metabolite quantification?

• Calibration standards are prepared in neat solvent only
• Standards are prepared in the same biological matrix as study samples to compensate for matrix effects
• Calibration uses internal standard only with no external standards
• Calibration curves are run on a different instrument to validate transferability

Correct Answer: Standards are prepared in the same biological matrix as study samples to compensate for matrix effects

Q10. Which evaporative concentration technique is preferred to minimize loss of volatile metabolites after extraction?

• Heating to 60–80°C under vacuum
• Nitrogen blowdown at low temperature with water bath cooling
• Lyophilization of organic extract at room temperature
• SpeedVac with high centrifugal force at ambient temperature

Correct Answer: Nitrogen blowdown at low temperature with water bath cooling

Q11. What is the most appropriate approach to handle small sample volumes (e.g., pediatric plasma) while ensuring adequate metabolite recovery?

• Scale down extraction volumes and use micro‑SPE or MEPS to maintain concentration and recovery
• Pool samples without regard to study design to increase volume
• Increase dilution during protein precipitation to increase recovery
• Perform direct injection of the tiny volume without cleanup

Correct Answer: Scale down extraction volumes and use micro‑SPE or MEPS to maintain concentration and recovery

Q12. During method development for metabolite identification by HRMS, why is minimal derivatization favored for LC‑HRMS workflows?

• Derivatization increases ion suppression universally
• Derivatization complicates exact mass interpretation and may obscure native metabolite masses
• Derivatization is incompatible with all chromatographic columns
• Derivatization always increases fragmentation complexity beneficially

Correct Answer: Derivatization complicates exact mass interpretation and may obscure native metabolite masses

Q13. Which control sample is most important to assess artifactual metabolite formation during sample preparation?

• Blank solvent injected into the LC-MS
• Process blank prepared by carrying a blank matrix through the full preparation procedure
• Calibration standard in neat solvent
• Quality control sample stored at −80°C

Correct Answer: Process blank prepared by carrying a blank matrix through the full preparation procedure

Q14. Which SPE sorbent is generally preferred when isolating moderately polar acidic metabolites from plasma?

• Reversed‑phase C18 under neutral conditions
• Strong anion exchange (SAX) or mixed‑mode anion exchange
• Normal‑phase silica without conditioning
• Silver‑impregnated silica for polar interactions

Correct Answer: Strong anion exchange (SAX) or mixed‑mode anion exchange

Q15. Why is it important to evaluate freeze‑thaw stability during metabolite sample preparation planning?

• To check if repeated freezing and thawing causes enzymatic conversion, degradation, or adsorption that alters metabolite levels
• To ensure sample color remains constant during storage
• To maximize protein precipitation efficiency
• To determine chromatographic retention time shifts

Correct Answer: To check if repeated freezing and thawing causes enzymatic conversion, degradation, or adsorption that alters metabolite levels

Q16. In metabolite profiling for non‑clinical studies, which practice improves detection of low‑abundance metabolites while reducing sample complexity?

• Use of high sample dilution before injection
• Fractionation (e.g., polarity or size‑based) prior to LC‑MS analysis
• Only analyzing intact tissue without homogenization
• Excluding internal standards to simplify spectra

Correct Answer: Fractionation (e.g., polarity or size‑based) prior to LC‑MS analysis

Q17. Which factor should be optimized to reduce carryover when analyzing metabolite-rich samples on LC‑MS?

• Increase sample injection volume indefinitely
• Optimize wash solvents and needle wash cycles, and use appropriate organic strength in wash
• Use only aqueous wash solvents between injections
• Decrease column flow rate to near zero between runs

Correct Answer: Optimize wash solvents and needle wash cycles, and use appropriate organic strength in wash

Q18. For GC‑MS metabolite analysis, what sample prep step is essential for thermally labile polar metabolites to improve volatility and stability?

• Direct injection of aqueous extract into GC inlet
• Derivatization (e.g., silylation with BSTFA) to form volatile derivatives
• SPE with C18 to remove all polar compounds
• Use of strong acids to protonate metabolites

Correct Answer: Derivatization (e.g., silylation with BSTFA) to form volatile derivatives

Q19. Which statement best describes the use of surrogate matrices in metabolite quantification?

• Surrogate matrices are acceptable when the native matrix is unavailable, but must be demonstrated equivalent in terms of matrix effects and recovery
• Surrogate matrices always give identical results to real matrices without testing
• Surrogate matrices refer to using water as the biological matrix
• Surrogate matrices are only used for qualitative studies and never for quantification

Correct Answer: Surrogate matrices are acceptable when the native matrix is unavailable, but must be demonstrated equivalent in terms of matrix effects and recovery

Q20. Which quality control measure during sample prep ensures consistent recovery across batches in metabolite studies?

• Running a single calibration curve at the start of the study and never rechecking
• Including low, medium and high QC samples prepared in the study matrix and processed with each batch
• Using different internal standards for each batch to compensate for variability
• Skipping centrifugation steps to save time

Correct Answer: Including low, medium and high QC samples prepared in the study matrix and processed with each batch

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