How is the Validation of Analytical Methods Performed?

The validation of analytical methods is an essential element of Good Manufacturing Practice (GMP). It provides documented evidence that an analytical test procedure is suitable for its intended purpose and, under defined conditions, generates reliable, reproducible, and robust results.

Analytical methods are used in the pharmaceutical industry, among other purposes, for the testing of starting materials, active substances (APIs), intermediates, and finished medicinal products. An inadequately validated method may lead to erroneous test results and thus represents a significant risk to product quality, patient safety, and regulatory compliance.

Regulatory Framework

In the EU GMP Guide Part I, validation of analytical methods is cited multiple times as a fundamental requirement. Chapter 1.9 already requires that test methods be validated. Chapter 2.8 assigns to the Head of Quality Control, among other responsibilities, the duty to ensure that the necessary validations are carried out. Chapter 6.15 explicitly states:

• "Testing methods should be validated. A laboratory that is using a testing method and which did not perform the original validation, should verify the appropriateness of the testing method. All testing operations described in the marketing authorisation or technical dossier should be carried out according to the approved methods."

For manufacturers of active substances, the EU GMP Guide Part II further specifies the requirements. Section 12.8 (Validation of Analytical Methods) states:

• "12.80 Analytical methods should be validated unless the method employed is included in the relevant pharmacopoeia or other recognised standard reference. The suitability of all testing methods used should nonetheless be verified under actual conditions of use and documented."
• "12.81 Methods should be validated to include consideration of characteristics included within the ICH guidelines on validation of analytical methods. The degree of analytical validation performed should reflect the purpose of the analysis and the stage of the API production process."
• "12.82 Appropriate qualification of analytical equipment should be considered before starting validation of analytical methods."
• "12.83 Complete records should be maintained of any modification of a validated analytical method. Such records should include the reason for the modification and appropriate data to verify that the modification produces results that are as accurate and
  reliable as the established method."

In addition to EU GMP requirements, national legislation may impose further binding obligations. For example, in Germany the Medicinal Products and Active Pharmaceutical Ingredients Manufacturing Ordinance (Arzneimittel- und Wirkstoffherstellungsverordnung (AMWHV)) requires that testing procedures be validated in accordance with the current state of science and technology and that critical test procedures be periodically evaluated and, where necessary, revalidated. Comparable country-specific regulations may apply in other jurisdictions.

In the United States, the requirements arise from 21 CFR Part 211. Section 211.165(e) provides:

• "The accuracy, sensitivity, specificity, and reproducibility of test methods employed by the firm shall be established and documented."

The requirements of the USP, in particular General Chapter <1225>, must also be taken into consideration.

Validation Parameters According to ICH Q2(R2)

The ICH guideline Q2(R2) “Validation of Analytical Procedures” represents the internationally harmonised standard for the validation of analytical methods and has replaced the previous version Q2(R1). ICH Q2(R2) is closely linked to ICH Q14 “Analytical Procedure Development.” While Q14 describes the systematic development of analytical procedures, Q2(R2) defines the requirements for their validation.

Although ICH Q2(R2) and ICH Q14 are not laws or regulations, they are regarded as reflecting the recognised state of science and technology.

Depending on the type of method, the following performance characteristics are relevant according to ICH Q2(R2):

• Specificity/Selectivity: "Specificity and selectivity are both terms to describe the extent to which other substances interfere with the determination of an analyte according to a given analytical procedure. Specificity is typically used to describe the ultimate state, measuring unequivocally a desired analyte. Selectivity is a relative term to describe the extent to which particular analytes in mixtures or matrices can be measured without interferences from other components with similar behaviour."
• Range: "The range of an analytical procedure is the interval between the lowest and the highest results in which the analytical procedure has a suitable level of precision, accuracy and response."
• Accuracy: "The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or as an accepted reference value and the value or set of values measured."
• Precision: "The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple samplings of the same homogeneous sample under the prescribed conditions. Precision can be considered at three levels: repeatability, intermediate precision and reproducibility. The precision of an analytical procedure is usually expressed as the variance, standard deviation or coefficient of variation of a series of measurements."
• Robustness: "The robustness of an analytical procedure is a measure of its capacity to meet the expected performance criteria during normal use. Robustness is tested by deliberate variations of analytical procedure parameters."

ICH Q2(R2) places greater emphasis than its predecessor on the fact that not all validation characteristics are universally required. Selection and extent must be scientifically justified and documented.

Implementation in Practice

The development of analytical procedures in accordance with ICH Q14 is based on a systematic understanding of:

• the analyte,
• the product matrix,
• critical method parameters.

Validation is conducted on the basis of a validation protocol, which defines, inter alia:

• the purpose and intended use of the method,
• the validation characteristics to be evaluated,
• acceptance criteria,
• statistical evaluation methods,
• responsibilities.

Validation is performed through predefined experimental studies. All activities must be documented in compliance with GMP requirements. Deviations must be assessed, root causes analysed, and, where appropriate, corrective and preventive actions (CAPA) initiated.

The validation report summarises the results and includes:

• an evaluation of each validation characteristic,
• an overall assessment of method performance,
• a clear statement regarding the suitability of the method for its intended purpose.

Only after formal approval may the method be implemented for routine use.

Revalidation is required, for example, if:

• significant changes are made to the method,
• new equipment or software is introduced,
• the product or matrix changes,
• trends, OOS (Out-of-Specification), or OOT (Out-of-Trend) results raise doubts about method performance.

Here, too, a risk-based approach in accordance with the pharmaceutical quality system must be applied.

Training, Working Groups and Further Information

For professional exchange and further interpretation, scientific conferences that discuss new regulatory developments, such as the annual PharmaLab conference held each November, are appropriate forums.

Both CONCEPT Heidelberg and the ECA also regularly offer training courses and seminars on analytical method validation.