This document provides a detailed overview of analytical method validation, covering performance parameters, validation steps, method capability, specificity, linearity, sensitivity, ruggedness, method transfer, stability-indicating assays, and compendial method validation.

Performance Parameters Required for Assay Validation

(Ref. USP 25 and ICH Q2A/B)

The following performance parameters are typically required for assay validation, though some may depend on the specific test:

• Method precision
• Method accuracy
• Method selectivity/specificity and system suitability
• Method linearity and range
• Method sensitivity
• Method ruggedness and robustness

Note: Lack of specificity may be compensated for with additional tests.

Steps of Analytical Method Validation

The following are fundamental steps in analytical method validation. However, individual validation projects may require unique steps based on the test method, laboratory conditions, equipment, sample criteria, reference standards, controls, and method source.

Step 1: Analytical Method Validation Protocol

Planning the validation scope, objective, methodology, and outcome is crucial. A validation protocol serves as the blueprint for all validation activities, ensuring proper documentation and reporting. A typical protocol includes:

• Statement of protocol scope
• Responsibilities (approval, execution, review)
• Required materials and instruments
• Test method statement (final draft)
• Experimental design details (for each parameter)
• Data recording documents and forms
• Acceptance criteria (for each parameter)

Step 2: Method Precision

Precision is the closeness of results to each other. At least six preparations should be analyzed, and results compared. Precision is measured in three ways:

• Repeatability: Precision of the operating system.
• Intermediate Precision: Precision of the analytical method.
• Reproducibility: Precision between analysts, days, and equipment.

Precision acceptance is based on pre-defined criteria relative to the method and product specification. Ideally, chromatographic method repeatability should be <1.0%. Suggested precision acceptance limits vary by sample type. (See table for categorized limits).

Step 3: Method Accuracy

Accuracy is how close measured results are to the actual amount present. Accuracy can be determined by:

• Spiking the active into a placebo matrix (25% to 150% of dose strength).
• Standard addition of active to the drug matrix.
• Direct comparison with an equivalent alternate method.

Sample stability should be considered, as degradation can affect accuracy. This applies to standards and samples standing for at least 24 hours.

Accuracy Calculation:

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

Accuracy can also be expressed as “bias” (e.g., -1.2% bias). Suggested accuracy acceptance limits are categorized by sample type. (See table).

Method Capability

Capability measures the method’s ability to consistently produce results within specification. It depends on:

• Specification width
• Method accuracy
• Method precision

Capability Calculation:

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

Step 4: Method Specificity (or Selectivity)

Specificity is the method’s ability to accurately and precisely measure the active analyte in the presence of interferences (impurities, degradants, excipients, solvents, drug matrix). For HPLC or GC assays, the ability to separate interferences indicates specificity. Specificity verification is important for stability-indicating assays.

Specificity Check:

Add the analyte to each potential interfering compound and assess:

• Ideally, no peaks in the mobile phase or diluent chromatogram (or elute with the solvent front).
• If peaks are present, they should elute at RRTs different from the API, internal standard, or impurity peaks.
• Other peaks should elute at RRTs different from other peaks in the chromatogram.
• Excipient peaks (fresh and degraded) should elute at RRTs different from the API, internal standard, or impurity peaks.

System Suitability for HPLC Methods:

System suitability determines instrument performance (sensitivity, retention) by analyzing a reference standard before the analytical batch. (See separate resource for more on System Suitability for HPLC analysis).

Step 5: Method Linearity and Range

Linearity is the method’s ability to produce test results proportional to analyte concentration within a given range. The range is the interval between upper and lower concentrations demonstrated to be accurate, precise, and linear.

Test at least five different concentrations in triplicate to establish linearity and range. Concentrations can be prepared by weighing and transferring, or by dilutions from a stock solution.

Linear regression analysis demonstrates linearity. The range is typically 50%-125% of the target active concentration. Degradant concentrations are evaluated separately. The correlation coefficient (r) should be >0.99 for the selected range (linear slope). The method should have a linear response over at least 75-125% of the target analyte concentration.

Step 6: Assay Sensitivity, LOD, and LOQ

Sensitivity is the response per unit of analyte. Two factors verify sensitivity:

• Limit of Detection (LOD): Lowest detectable concentration (not necessarily quantifiable). A parameter of limit tests.
• Limit of Quantitation (LOQ): Lowest quantifiable concentration with acceptable precision and accuracy. A parameter of quantitative impurity assays.

LOD and LOQ Determination:

• Evaluate detection for LOD or accuracy and precision at the LOQ.
• Evaluate signal-to-noise ratio (for instrumental methods).
• Precision of response and slope: LOQ = (10xσ)/b, LOD=(3.3xσ)/b (σ = standard deviation of response, b = slope).

There are no specific LOD/LOQ acceptance criteria. For active APIs:

• LOQ should be ≤0.5% (preferably lower).
• LOD should be ≤0.25% (preferably lower).
• Precision should be 5-10% at the LOQ.
• LOD signal-to-noise ratio should be 3:1.
• LOQ signal-to-noise ratio should be 10:1.

(Information about additional GMP resources omitted)

Step 7: Method Ruggedness and Robustness

Ruggedness/Robustness is the reproducibility of results under various normal test conditions. Ruggedness refers to external factors, while robustness refers to internal factors.

Ruggedness/Robustness is determined by measuring accuracy and precision under various conditions:

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

If ruggedness studies identify influential factors, they should be controlled by the test method.

Analytical Method Validation for Method Transfer

Analytical method validation is strongly recommended during method transfer to another laboratory (e.g., R&D to QC). It ensures the method is robust enough for transfer and generates evidence that transferred methods perform equivalently under changed conditions.

Method transfer requires documented evidence (protocol and procedure) verifying:

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

The transfer protocol qualifies the new laboratory, trains staff, and ensures equivalent performance.

Analytical Method Transfer Requirements:

• Detailed Method: Unambiguous procedure with example chromatograms and system suitability calculations.
• Method Development Report: Reviews method development and justifies parameter choices.
• Transfer Protocol: Details requirements, timing, responsibilities, acceptance criteria.
• Validation Report: Summarizes validation activities and results.

Stability-Indicating Assay Requirements:

Stability-indicating assay validation must verify the method’s stability-indicating nature. There should be a defined degradation pathway. The method should:

• Be selective for degradants
• Be robust to interference
• Chromatographically separate retention times
• Have a mass-balance >90%

Compendial (Pharmacopoeial) Test Validation:

Compendial methods (e.g., from EP, USP) are generally considered reliable under usual conditions. However, each laboratory’s conditions, equipment, and product applications differ. Therefore, compendial methods must be verified for suitability under “actual conditions of use” in each laboratory.

Conclusion

Reliable analytical method validation is a fundamental GLP requirement, essential for product registration and GMP inspections. It provides evidence of robust and reproducible test methods, ensuring product safety, purity, effectiveness, and traceability. A planned approach, including a protocol, specifications, sequential validation steps, and reporting, is crucial.

Key considerations during analytical method validation include:

• Performance parameters and acceptance criteria.
• Analytical method validation protocol components.
• Inter-laboratory method transfer requirements.
• Stability-indicating assay requirements.
• Compendial method validation.