Below is a simplified overview of the key validation parameters. Always consult your Regulatory Affairs team for specific requirements.

1. Accuracy

• Measures how close your results are to the true value.
• Demonstrated through:
  • Comparison to a certified reference standard
  • Percent recovery studies
  • Standard addition experiments

2. Precision

Defined by the International Council for Harmonisation (ICH) in three levels:

• Repeatability: Same analyst, same instrument, short time frame.
• Intermediate Precision: Different days, analysts, instruments.
• Reproducibility: Across different laboratories (often during method transfer).

3. Linearity

• Assesses how well the detector response correlates with analyte concentration.
• Typically evaluated using:
  • A calibration curve
  • Correlation coefficient (R²)
  • Y-intercept and slope

4. Range

• The span between the lowest and highest concentrations where the method is accurate, precise, and linear.
• Must cover expected sample concentrations.

5. Limit of Detection (LOD) & Limit of Quantitation (LOQ)

• LOD: Lowest concentration that can be reliably detected (S/N ≈ 3).
• LOQ: Lowest concentration that can be reliably quantified (S/N ≈ 10).
• Can also be calculated using:
  • LOD = 3.3 S0/b
  • LOQ = 10 S0/b
    • where ( S_0 ) is the standard deviation of the response and ( b ) is the slope of the calibration curve.

6. Specificity

• The method’s ability to distinguish the analyte from other components in the matrix.
• Demonstrated through:
  • Chromatographic separation
  • Forced degradation studies
  • Orthogonal methods
  • Selective detection (e.g., MS or immunoassays)

7. Ruggedness

• Evaluates method consistency under variable conditions, such as:
  • Different analysts
  • Instrument models
  • Column lots
  • Solvent suppliers

8. Robustness

• Assesses how small, deliberate changes in method parameters affect results.
• Examples include:
  • Flow rate
  • Column temperature
  • Mobile phase pH
  • Gradient slope
• Results are compared to system suitability criteria (e.g., resolution, retention time).

9. Octanol-Water Partition Coefficient (Log P)

• Describes a compound’s hydrophobicity.
• Calculated as the ratio of concentrations in octanol vs. water at equilibrium.
• Useful for predicting:
  • Retention behavior in reversed-phase HPLC
  • Solubility and extraction efficiency

10. Recovery

• Measures how much of the analyte is recovered from the sample matrix after processing.
• Important for methods involving extraction or sample prep.