Analytical Method Validation (AMV)

Introduction

Analytical method validation is the documented process of demonstrating that an analytical procedure is suitable for its intended purpose. AMV ensures that an analytical method is suit for its intended purpose. This applies to all analytical tests carried out including those for identifying specific compounds, quantification of APIs, or identifying and/or quantifying impurities in a pharmaceutical product

From the definition, we can see that the process is documented, which means its rigorous planning, execution and comprehensive documentation are crucial as the technical performance of the method, such that in the end there will be an auditable, formal documentation of the analytical process

Analytical procedures, whether newly developed or revised, must be validated to include consideration of specific performance characteristics as outlined in ICH guidelines. These key performance characteristics are detailed in following sections

Key Performance Characteristics

Specificity/Selectivity

It is the ability of the analytical method to unequivocally assess the analyte of interest in the presence of various components in the mixture. A common acceptance criterion is that excipient compounds and potential impurities/degradants must not interfere with the analysis for accurate quantification of the targeted analyte i.e. absence of interference.

When a procedure cannot sufficiently distinguish analyte of interest, a combination of two or more procedures is recommended so as to reach level of selectivity required

ICH Q2(R2) introduces an important concept of orthogonal procedure comparison. This involves utilizing two or more separate analytical methods that employ different physicochemical principles or detection mechanisms to analyze and verify a particular parameter, thereby providing independent and complementary support for conclusions.

Accuracy

It is the closeness of agreement between measured value during an analytical procedure and the value which is accepted as either the conventional true value or the accepted reference value

Accuracy is verified by following studies

• Spiking studies: Involves preparing a matrix of compounds, with addition of known concentration of analyte (spiked sample) and then performing an analytical procedure for its evaluation and recovery
• Reference material comparison: results from an analytical procedure are compared to theoretically expected results
• Orthogonal procedure comparison: 2 or more analytical procedures which employ different measurement principles are used and their results are compared if they are in agreements

In ICH Q2 (R2) update, it promotes the approach of combining accuracy and precision evaluations. The allowance for a "combined approach" to evaluating accuracy and precision suggests a more efficient and statistically integrated assessment of method performance, since these two parameters are inherently interdependency.

While a method can be precise but inaccurate (consistently yielding results that are wrong but close to each other), or accurate but imprecise (yielding results that are scattered but centered on the true value), a truly reliable method requires both.

Precision

It is the closeness of agreement between a series of measurements obtained from multiple samplings of same homogenous sample under prescribed conditions. Precision should ideally be investigated using homogeneous, authentic samples.

However, if obtaining a homogeneous authentic sample is not feasible, it may be investigated using artificially prepared samples or a sample solution. Precision is considered at 3 levels which are

• Repeatability: This assesses precision under the same operating conditions over a short interval of time, involving the same analyst, same equipment, and within the same laboratory.
• Intermediate Precision: This evaluates precision within the same laboratory but under varying conditions, such as different days, different analysts, or different pieces of equipment.
• Reproducibility: Involves inter-laboratory trial. It is not a prerequisite for regulatory submission

 The assessment of precision at various levels i.e. repeatability, intermediate precision & reproducibility is a sophisticated approach that directly addresses the real-world variability inherent in analytical measurements within a pharmaceutical environment.

This structured evaluation ensures that a method is not only consistent within a single analyst's hands but also sufficiently robust to yield reliable results across different operational conditions, different personnel, and even different laboratories.

 This comprehensive assessment is paramount for successful method transfer between sites, ensuring global manufacturing consistency, and maintaining confidence in batch release data and stability study results.

Linearity

It is the method's ability to obtain test results that are directly proportional to the concentration (amount) of the analyte in the sample, within a given defined range. To assess linearity during validation, a minimum of five concentrations appropriately distributed across the range is recommended. The results are then recorded, and a plot of concentration (x-axis) versus mean response (y-axis) is generated.

Statistical calculations, including the regression equation and coefficient of determination (r²), are performed. A common acceptance criterion for linearity is a correlation coefficient (r²) of ≥ 0.999 for the range of 80% to 120% of the target concentration. Additionally, the y-intercept should typically be ≤ 2% of the target concentration response.

Range

It is defined as the lower and upper limit concentration of the analyte to which an analytical procedure is shown to demonstrate precision, accuracy and linearity attributes

It explicitly delineates the operational limits within which the method is proven to be reliable and fit for purpose, thereby preventing its inadvertent or deliberate misuse outside of its scientifically established and regulatory-accepted conditions

Lower Range Limits

Lower range limits include Detection Limit (DL/LOD) and Quantification Limit (QL/LOQ)

Detection Limit is the lowest amount of an analyte which can be detected during an analytical procedure.

Quantification Limit is the minimum amount of an analyte which can be quantified with suitable precision and accuracy

Lower range limits can be determined using some of the following methods:

1. Signal-to-noise ratio: Determined by comparing measured signals form a known low concentration of an analyte with that of a blank sample. This method works on analytical procedures which exhibit baseline noise.

For DL, signal-to-noise ratio of 3:1 is considered acceptable

For QL. Signal to noise ratio of 10:1 is considered acceptable

2. Accuracy & Precision at lower range limits: Accuracy and precision measurements are done at low concentration levels of analyte to validate the QL and DL

The integration of LOD and LOQ in ICH Q2 (R2) into “lower range limits” helps simplify the validation process, reduces redundant reporting and focuses the analytical procedure more on the lowest possible point to which reliable quantitative results can be obtained, which is most critical aspect in impurity testing.

Robustness

It is the capacity of an analytical procedure to remain unaffected by small, deliberate change variations in procedure’s parameters. This is performed by intentionally introducing minor adjustments to the analytical procedure parameters and evaluating any response to the variation done

Robustness ensures that an analytical procedure will remain stable in the event of minor variation during normal usage, which minimizes out-of-specification results

Analytical Procedure Validation Protocol

This serves as the blueprint to the analytical method validation study.

Like any protocol, it must have the purpose and objectives of the analytical procedure. All the necessary performance characteristics to be validated must be stated according to ICH Q2 R2 guidelines.

The report's structure and content are designed to provide transparency and traceability, allowing auditors and stakeholders to fully understand the method's capabilities and limitations.

Analytical Procedure Validation Report

It is a comprehensive record of the validation study, documenting its findings and conclusions. These results must be compared directly to the predefined Performance Criteria established in the protocol

The report must conclude with a definitive statement regarding the suitability of the analytical procedure for its intended use. The validation report is the culmination of the entire validation process and serves as the documented evidence required for regulatory submissions and ongoing quality assurance. This is essential in maintaining product quality throughout the product lifecycle and ultimately safeguarding patient safety

Conclusion

AMV is a regulatory requirement globally, which is essential to maintaining drug quality. Guidelines such as the ICH Q(2) and its alignment with ICH Q14 serves as international benchmarks for analytical procedure validation, which promotes harmonization and streamlining global drug development and registration