Bioanalytical method validation – USFDA guidelines MCQs With Answer

Bioanalytical method validation – USFDA guidelines MCQs With Answer

Introduction: This quiz collection covers key principles of bioanalytical method validation as described by USFDA guidance, tailored for M.Pharm students studying Modern Bio-Analytical Techniques (MPA 202T). The questions focus on fundamental validation parameters — accuracy, precision, selectivity, sensitivity (LLOQ/LOD), calibration, matrix effects, stability, dilution integrity, carryover, system suitability, internal standards, incurred sample reanalysis and acceptance criteria. Each MCQ challenges understanding of regulatory expectations and practical laboratory application, helping you prepare for exams and real-world bioanalytical method development and validation. Use these items to test comprehension and reinforce best practices required for GLP-compliant bioanalysis.

Q1. What is the USFDA definition of bioanalytical method validation?

• A formal process to document laboratory equipment maintenance schedules
• A set of experiments to establish the suitability of a method for its intended purpose by assessing performance characteristics such as accuracy, precision, selectivity and stability
• A process to optimize chromatographic separation only
• A requirement to test only the limits of detection and quantitation

Correct Answer: A set of experiments to establish the suitability of a method for its intended purpose by assessing performance characteristics such as accuracy, precision, selectivity and stability

Q2. Which of the following lists contains the primary validation parameters required by USFDA guidance?

• Accuracy, precision, selectivity/specificity, sensitivity (LLOQ), calibration curve, recovery, stability, matrix effect, dilution integrity
• Shelf-life, packaging integrity, microbial limits, endotoxin levels
• Only limit of detection (LOD) and limit of quantitation (LOQ)
• Instrument warranty, operator training records, SOP approvals

Correct Answer: Accuracy, precision, selectivity/specificity, sensitivity (LLOQ), calibration curve, recovery, stability, matrix effect, dilution integrity

Q3. What are the typical USFDA acceptance criteria for accuracy and precision for QC samples (excluding LLOQ)?

• Accuracy within ±15% of nominal and precision (%CV) ≤15%
• Accuracy within ±5% and precision ≤5%
• Accuracy ±30% and precision ≤30%
• Accuracy ±50% and precision ≤50%

Correct Answer: Accuracy within ±15% of nominal and precision (%CV) ≤15%

Q4. How does USFDA define the Lower Limit of Quantitation (LLOQ)?

• The highest concentration that can be measured with perfect accuracy
• The lowest concentration that can be measured with acceptable accuracy and precision (typically ±20% at LLOQ)
• The concentration equal to the mean of the calibration standards
• A theoretical value based only on signal-to-noise ratio

Correct Answer: The lowest concentration that can be measured with acceptable accuracy and precision (typically ±20% at LLOQ)

Q5. How many non-zero calibration standards does USFDA guidance recommend as a minimum for a calibration curve?

• At least two non-zero calibrators
• At least six non-zero calibration standards (plus blank/zero as appropriate)
• Exactly ten calibration standards only
• No calibration standards are required if using internal standardization

Correct Answer: At least six non-zero calibration standards (plus blank/zero as appropriate)

Q6. What is the matrix effect in LC-MS/MS bioanalysis?

• A change in analyte response due to co-eluting components from biological matrix affecting ionization or signal
• The variation in chromatographic column packing
• The dissolution rate of the analyte in buffer
• An intentional addition of surfactant to the sample

Correct Answer: A change in analyte response due to co-eluting components from biological matrix affecting ionization or signal

Q7. What does USFDA recommend for incurred sample reanalysis (ISR)?

• ISR is not necessary for regulatory studies
• Reanalyse at least 10% of study samples, with acceptance that at least two-thirds of ISR results should be within ±20% of original values
• Reanalyse all samples in duplicate and accept any difference
• ISR should be performed only if the initial run fails system suitability

Correct Answer: Reanalyse at least 10% of study samples, with acceptance that at least two-thirds of ISR results should be within ±20% of original values

Q8. Which stability experiments are commonly required during bioanalytical method validation?

• Bench-top (short-term), autosampler, freeze–thaw, long-term (storage) and stock solution stability
• Only long-term stability at room temperature
• Only stability in presence of strong acids
• No stability testing is required for LC-MS/MS methods

Correct Answer: Bench-top (short-term), autosampler, freeze–thaw, long-term (storage) and stock solution stability

Q9. How should carryover be assessed and controlled according to common USFDA-based practice?

• Ignore carryover; it is not important for quantitation
• Inject a blank after the upper limit of quantitation standard and ensure analyte response in blank is ≤20% of the LLOQ and internal standard ≤5% of its typical response
• Only use larger injection volumes to dilute carryover
• Replace columns after every injection to avoid carryover

Correct Answer: Inject a blank after the upper limit of quantitation standard and ensure analyte response in blank is ≤20% of the LLOQ and internal standard ≤5% of its typical response

Q10. When constructing calibration curves, which weighting strategy is commonly used to handle heteroscedastic data?

• No weighting at all, always use unweighted regression
• Use 1/x or 1/x^2 weighting when heteroscedasticity is present and justify the choice
• Use logarithmic weighting exclusively
• Discard low concentration points to avoid heteroscedasticity

Correct Answer: Use 1/x or 1/x^2 weighting when heteroscedasticity is present and justify the choice

Q11. What is the purpose of demonstrating dilution integrity?

• To test sample color change after dilution
• To confirm that samples above the upper limit can be accurately measured after validated dilution without bias or loss of precision
• To establish the LOD
• To validate autosampler needle wash solutions only

Correct Answer: To confirm that samples above the upper limit can be accurately measured after validated dilution without bias or loss of precision

Q12. Which parameters are typically part of system suitability testing for chromatographic bioanalysis?

• Column theoretical plates, peak symmetry/tailing factor, retention time repeatability, resolution between peaks
• Microbial load and endotoxin level of solvents
• Operator handwriting legibility
• Number of calibration standards only

Correct Answer: Column theoretical plates, peak symmetry/tailing factor, retention time repeatability, resolution between peaks

Q13. What is the preferred type of internal standard for quantitative LC-MS/MS bioanalysis according to best practice?

• A structurally unrelated compound with different retention time
• A stable isotope-labeled analog of the analyte (e.g., deuterated or 13C-labeled)
• Any compound that fluoresces under UV
• Plain solvent without analyte

Correct Answer: A stable isotope-labeled analog of the analyte (e.g., deuterated or 13C-labeled)

Q14. How many QC levels and replicates are generally recommended for method validation runs?

• At least one QC level with two replicates
• At least four QC levels (LLOQ, low, mid, high) with a minimum of five replicates per level across validation runs
• Ten QC levels with one replicate each
• No QCs are necessary if calibration curve fits well

Correct Answer: At least four QC levels (LLOQ, low, mid, high) with a minimum of five replicates per level across validation runs

Q15. What is the acceptance criterion for back-calculated concentrations of calibration standards?

• All calibrators must match nominal exactly (0% error)
• Within ±15% of nominal for non-LLOQ calibrators and within ±20% for LLOQ calibrator
• Within ±50% for all calibrators
• Calibrators do not need acceptance criteria

Correct Answer: Within ±15% of nominal for non-LLOQ calibrators and within ±20% for LLOQ calibrator

Q16. How does USFDA guidance distinguish selectivity from specificity in bioanalytical methods?

• Selectivity is the method’s ability to quantify the analyte in presence of other components (i.e., absence of interferences); specificity is often used interchangeably but selectivity is preferred term
• Selectivity refers to instrument sensitivity only
• Specificity is about sample storage conditions while selectivity is about column choice
• They are completely unrelated concepts in bioanalysis

Correct Answer: Selectivity is the method’s ability to quantify the analyte in presence of other components (i.e., absence of interferences); specificity is often used interchangeably but selectivity is preferred term

Q17. How is accuracy usually expressed in bioanalytical method validation?

• As the absolute difference in mV signal
• As %Bias = ((measured concentration − nominal concentration)/nominal concentration) × 100
• Only as units of concentration without normalization
• As the instrument serial number

Correct Answer: As %Bias = ((measured concentration − nominal concentration)/nominal concentration) × 100

Q18. Which components constitute precision in the context of USFDA bioanalytical guidance?

• Repeatability (intra-day) and intermediate precision (inter-day, different analysts, instruments)
• Only the variation between different instruments in different labs
• Only the theoretical calculation based on detector sensitivity
• Precision refers to the method’s speed to process samples

Correct Answer: Repeatability (intra-day) and intermediate precision (inter-day, different analysts, instruments)

Q19. How many independent validation runs are typically required to demonstrate method reproducibility?

• Only one run is sufficient if QC passes
• At least three independent validation runs on separate days
• Exactly ten runs on the same day
• No runs required if calibration curve R2 > 0.999

Correct Answer: At least three independent validation runs on separate days

Q20. When should incurred sample reanalysis samples be selected and what acceptance is expected?

• Select ISR samples only from quality control material; acceptance is arbitrary
• Select ISR from actual study samples (representative concentration range, at least 10% of study samples) and expect most pairs (e.g., two-thirds) to agree within ±20%
• ISR must be performed only on placebo samples
• Reanalyse all samples twice and accept any variability

Correct Answer: Select ISR from actual study samples (representative concentration range, at least 10% of study samples) and expect most pairs (e.g., two-thirds) to agree within ±20%

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