Method Validation

Method Validation is the process used to evaluate whether or not a specific analytical procedure is suitable for its intended use.


The results from a method validation can be used to ascertain the accuracy, specificity, and reliability of the analytical data.


Analytical methods should be validated or re-validated

  • before being used on a routine basis

  • whenever conditions change under which the method was originally validated (for example: the method will be run on a different instrument with different capabilities or some aspect of the sample has changed such as different excipients in the formulation)

  • whenever the method itself is altered

The validity of an analytical procedure should be demonstrated through a specific series of tests using samples and/or USP reference standards similar to the material that will be routinely analyzed with the method.

Method validation parameters should include:

  • Accuracy - How close is the result to the correct answer?

Accuracy is typically determined using a minimum of nine determinations over a minimum of three concentration levels covering a specified range.

  • Precision - How close are the results of replicate measurements made on the same sample?

Precision is expressed as relative standard deviation of a series of measurements. Generally, the percent RSD should not be more than 2%.

  • Intermediate Precision - How do variations within the same laboratory affect the analytical procedure?

Intermediate precision shows how variations such as different days, analysts, or equipment affect the analytical procedure. The relative standard deviation and the confidence interval is reported.

  • Specificity - Are there any interference's? Are we measuring what we think we are measuring?

The method should be able to discriminate between closely related compounds that may be present in the sample such as impurities, degradation products, and matrix components. it must be demonstrated that the method is unaffected by spiked impurities and excipients. Specificity can be established by the resolution between two compounds that elute close together.

  • Working Range - What range of analyte concentrations (linearity) can be reliably measured?

Linearity is the ability of the method to obtain results that are directly proportional to the analyte concentration within a specified range. Typically, linearity is evaluated at a minimum of five concentrations.

For assay, the specified range is 80% - 120% of the targeted concentration.

The linearity relationship is established by statistical data obtained from the correlation coefficient, Y-intercept and the slope of the regression line.

What is the limit of detection?

The detection limit is the lowest concentration at which the analyte in a sample can be detected but not quantitated.

What is the limit of quantitation?

The quantitation limit is the lowest concentration at which the analyte in a sample that can be determined with precision and accuracy with the method.

Some of the approaches for determining the detection and quantitation limit include signal-to-noise ratio, standard deviation of the response and the slope.

  • Robustness - Do we have the control necessary to perform each stage of the procedure?

Robustness challenges how variations in operational parameters impact the method. The variations include changes in temperature, flow rate and solvent composition.

Stability indicating methods also include forced degradation studies and solution stability testing.

The preparation and execution of the method validation should follow a detailed step-by-step validation protocol. All data must be recorded in real time as the validation is executed.

Method Validation

  • should be extensive enough as necessary to ensure that it is suitable for the proposed application (fit-for-purpose)

  • should be customized to be relevant to the customer's needs

BRS Analytical adheres to the guidance provided in the USP General Chapter <1225> Validation of Compendial Methods.

 
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