Analytical Method Validation for Quality Control in GMP

Basic validation philosophy

Establishing documented evidence which provides a high degree of assurance that a specific process (analytical test method) will consistently produce a product (assay result) meeting its predetermined specifications and quality attributes (accuracy, precision, etc.) FDA – Process Validation Guidelines (1987)

Pre-requisites for method validation

In order to validate a test method, there are some important pre-requisites:

  • Qualified instrumentation
  • Well-developed and understood documented method
  • Reliable, stable reference standard
  • Trained and “qualified” analysts
  • Method is robust to factors that may
  • Change or be uncontrolled
  • Well-documented method validation
  • Protocol and record system

Classes of analytical procedures and relevant protocols

We can classify different analytic methods into the following classes:

  • Tests for identity of the active
  • Qualitative measurements for impurities/degradants
  • Limit tests for impurities/degradants
  • Quantitative assays of an API or drug product active material
  • Physical product characteristics, e.g. dissolution rate

Components of validation protocol

Sections of a typical protocol:

  • Statement of protocol scope
  • Responsibilities for approval, execution, and final review
  • List of required materials and instruments
  • Statement of the test method (in final draft)
  • Details of the experimental design for verifying each performance parameter
  • Documents and forms for recording validation results
  • Acceptance criteria for each performance parameter

Method performance parameters

To successfully complete an analytical method validation, you will need to validate to all or a combination of, these parameters:

  • Precision
  • Accuracy
  • Linearity and range
  • Selectivity/specificity
  • Sensitivity
  • Ruggedness and system suitability

Precision of the test method 

Precision:

The closeness of results with respect to each other.

To determine precision, a minimum of 6 preparations should be made, analyzed, and their results compared.

Precision is measured in three ways:

  • Repeatability – measures the precision of the operating system.
  • Intermediate precision – measures the precision of the analytical method.
  • Reproducibility – measures the precision between different analysts on different days and using different equipment.

We accept precision according to pre-defined criteria that are relative to the method itself and the product specification. Ideally, repeatability of chromatographic methods should be < 1.0%.

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Method accuracy

Accuracy:

How close the measured results of the test are to the actual amount that we know is present.

Ways to determine accuracy:

  1. Spike the active into a placebo matrix using amounts ranging from 25% to 150% of dose strength.
  2. Standard addition of active to the drug matrix.
  3. Direct comparison of two alternate methods for equivalence.

We should also consider prepared sample stability. If samples degrade, this may affect our accuracy. This check applies to standards and samples standing for a minimum of 24 hours on the bench.

Calculating Accuracy:

% Accuracy = 100 x [(Experimental amount – Theoretical amount)/Theoretical amount]

Accuracy may also be expressed as “bias” of the method, e.g. -1.2% bias.

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Method capability

Capability:

A measure of the method’s ability to consistently produce results that are within specification. The capability is dependent on:

  1. the specification width
  2. the accuracy of the method
  3. the precision of the method

Calculating Capability:

Cp method = [ (USL – LSL) – 2 x | average bias | ] / 6 x σ method

Cp > 2.5               Excellent capability

Cp 2.4 to 1.0        Good to poor capability

Cp < 1.0                Unacceptable

USL = Upper specification limit
LSL = Lower specification limit
bias = Accuracy

σ method = Intermediate precision 

Method specificity (or selectivity)

Specificity:

A method’s ability to accurately and precisely select or measure the active analyte in the presence of possibly interfering compounds such as impurities, degradants and the drug matrix/excipients.

For HPLC/GC assays, the ability of the method to separate interfering compounds is an indication of specificity.

Verification of specificity is important for methods used for stability-indicating assays.

How to check specificity:

Add the analyte to each of the potential interfering compounds, and assess its ability to meet the following:

  • Ideally, no peaks are present in the chromatogram of the mobile phase or diluents. If peaks are present, they elute with the solvent
  • If peaks are present, they should elute at RRTs that are not the same or similar to those of the API, internal standard, or any impurity peaks.
  • If any other peaks are present, they should also elute at RRTs that are not similar to those of any other peaks in the chromatogram.
  • All excipient peaks from both the fresh and degraded workups should elute at RRTs that ate not the same or similar to those of the API, internal standard, or impurity peaks.

Linearity and Range

Linearity:

The measure of the method’s ability to produce results that are proportional to the concentration of the analyte. Acceptance criteria should be r2>-0.99.

Linear regression analysis checks that linearity is acceptable.

The range refers to the range over which this proportionality occurs.
The working range should ideally be 50-150% but must meet 75-125%.

Assay sensitivity, LOD, and LOQ

Sensitivity:

The response obtained for a given amount of analyte. There are two factors that are used to verify sensitivity: the limit of detection (LOD) and the limit of quantitation (LOQ).

LOD: The lowest concentration that can be detected but not necessarily quantified. A parameter of limit tests.

LOQ: The lowest concentration that can be detected with acceptable precision and accuracy. A parameter of quantitative impurity assays.

Ways to determine LOD and LOQ:

  1. Evaluation of acceptable detection for LOD, or accuracy, and precision at the nominated LOQ.
  2. Evaluation of signal-to-noise ratio for instrumental methods that exhibit baseline signals.
  3. Precision of response and the slope where:
    LOQ = (10xσ)/b and LOD=(3.3xσ)/b

σ = standard deviation of the response
b = slope of the calibration curve

There are no specific acceptance criteria for LOD and LOQ.

The general aim for active APIs:

  • LOQ should not be more than 0.5%, and preferably much less
  • LOD should not be more than 0.25%, and preferably much less
  • Precision should be 5-10% at the defined limit for LOQ
  • LOD signal-to-noise ratio should be 3:1 at the defined limit
  • LOQ signal-to-noise ratio should be 10:1 at the defined limit

Ruggedness and Robustness

Ruggedness/Robustness:

The degree of reproducibility of test results obtained by the analysis of the same samples under a variety of normal test conditions. Ruggedness refers to external factors that can affect the method; robustness refers to internal factors that can affect the method.

Ruggedness or robustness can be determined by measuring the accuracy and precision under a variety of conditions, including:

  • Different days
  • Different analysts
  • Different laboratories
  • Different instruments
  • Different columns
  • Different reagent lots
  • Different assay temperatures
  • Different elapsed sample preparation/assay times

If ruggedness studies indicate that any factor is likely to influence the assay, then this should be controlled by the published test method.

Method Transfer Verification

Method transfer ensures that a validated method is robust enough to transfer to another laboratory, e.g. from the R&D to a QC laboratory.

Method transfer requires documented evidence (a protocol and procedure) that should verify the following attributes of the method:

  • Precision
  • Accuracy
  • Linearity/range
  • Ruggedness/robustness

The transfer protocol qualifies the new laboratory, trains its staff, and ensures that the new site can perform the method to the same standards as the originating site.

Requirements for method transfer

The following documentation is expected for a method transfer:

Detailed method: The procedure should be unambiguous with example chromatograms of the standard, a typical sample, and a system suitability limit with calculations.

Method development report: The report reviews the method development and provides justification for choice of operational parameters and conditions.

Transfer protocol: The protocol details requirements, timing, responsibilities, acceptance criteria, etc.

Validation report: The summary should include summary of validation activities and results.

Requirements of stability indicating assays

The validation of stability indicating assays needs to verify the stability indicating nature of the method.

There should be a defined degradation pathway for the active.

A stability indicating method should:

  • Be selective for degradants
  • Be robust to interference by product matrices
  • Be able to chromatographically separate retention times
  • Mass-balance account for >90% of contents

Compendial (pharmacopoeial) method validation

Compendial methods are those that come directly from pharmacopoeias. They are generally considered reliable under usual conditions of use.

Pharmacopoeial methods for finished dose should be validated.

Every laboratory has different conditions and equipment, and may apply the method in different ways to different products. Therefore, we cannot assume that a compendial method is validated. We need to verify its suitability under the “actual conditions of use” in our laboratory.

Key facts regarding analytical method validation

Reliable analytical methods (validation) is a fundamental GLP requirement. It is also important for product registration, and during GMP inspection of laboratories.

You should now be able to:

  • List performance parameters required and their acceptance criteria.
  • Identify what is involved in an analytical method validation protocol.
  • List requirements for an interlaboratory method transfer.
  • Understand the requirements of stability indicating assays.
  • Understand why compendial methods need to be validated.

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