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Your method validation questions answered

In the context of bioanalysis services, method validation ensures that the method is linear, reliable and reproducible and is sufficiently sensitive, stable, selective, accurate and precise to quantify the actual concentrations of analyte which will be present in the study samples.

Regulatory guidance for bioanalytical validation studies suggests the following validation parameters be defined in a protocol, study plan or SOP along with applicable acceptance criteria prior to execution of the experiments:

  • Selectivity
  • Matrix effects
  • Accuracy
  • Precision
  • Reproducibility
  • Sensitivity
  • Linearity and range
  • Lower limit of quantitation (LLOQ)
  • Upper limit of quantitation (ULOQ)
  • Stability evaluations (stock solution, freeze/thaw, short term matrix stability, whole blood stability and post-processed sample stability)
  • Effect of haemolysed / lipaemic samples and dilution evaluation

Method validation may involve a single analyte, multiple analytes, or a parent and metabolites. In any case, validation should be performed using the same matrix as targeted for study sample analysis. During method validation, a blank biological matrix will be spiked with the analyte(s) of interest using solutions of reference standard(s) to prepare calibration standards, QC and stability samples. In addition, suitable internal standard(s) is/are added during sample processing in chromatographic methods.

The three types of validation

  1. Full validation: Performed for new developed bioanalytical methods or when additional analytes or metabolites are added for quantitation
  2. Partial validation: Performed when modifications to an already validated bioanalytical method are performed. For example, a new detection system, change in sample matrix or calibration range
  3. Cross validation: A comparison of validation parameters when two or more bioanalytical methods are used to generate data within the same project. Cross validation may also be performed when data are generated using different analytical techniques eg. LC–MS/MS vs. ELISA, or where there are modifications in a validated method (different LC column, mobile phase, etc.)

Non-clinical methods (non-human species)

For non-clinical methods, a full validation is often performed first in the rodent due to the need for enhanced care regarding known enzymatic effects. A partial validation may be performed for the second species unless the method has been altered, and then a full validation is highly recommended.

Clinical methods (human)

For clinical methods, full validations are required. When validating between matrices ie. human plasma to human urine, a full validation is recommended to evaluate precision, accuracy, selectivity, and stability. For minor changes to a method within the same matrix and calibration range, a partial validation may be performed. For example, if the internal standard for a plasma assay that has been fully validated is changed, a full validation is not required as ancillary experiments that are independent of IS identity, such as matrix stability, are not affected by a change in internal standard.

Ancillary experiments that are impacted by IS identity, such as reinjection reproducibility or extract stability, must be repeated. The changes being made to the method must be evaluated to determine which experiments should be repeated during the partial validation, keeping in mind the purpose of each experiment and the effect the change would have on each experiment.