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September 1, 2023 Dr Pramod Kr Pandey
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Validation

Ultimate Guide in Analytical Method Validation (AMV)

Table of contents

Analytical Method Validation: Introduction and Outcome

Analytical method validation (AMV) decides the reliability of an Analytical method for the intended use. Since analytical method determines the quality of the Drug substances and Drug Products and that is why Analytical method validation plays a vital role in any pharmaceutical industry. It is expensive, time – consuming and also requires additional resources. Moreover, it requires knowledge and expertise. That’s why I decided to share skill-based knowledge on this. In this article, I will discuss analytical validation step by step in a simple and easy manner. I will cover guidelines on analytical validation, a 7-step strategy for effective Analytical method validation, 6-classification of the chromatographic methods, 11 most important Analytical Method Validation Parameters, Validation protocol and validation report. I will also cover various applications of Analytical method validation.

Analytical Method Validation

Establishing documentary evidence that provides a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications and quality characteristics is called Analytical Method validation, or AMV. Validation includes both equipment and method.

Drug substances that need validation may be final Active Pharmaceutical Ingredients (APIs), Intermediates, Raw materials and Final products. *Generally validation is performed for the following types of methods:

  • Chromatographic method (e.g. HPLC, GC,TLC)
  • Spectroscopic method (e.g. UV and FTIR)
  • Titration method
  • Mass spectroscopic method (LC-MS, GC-MS and ICP-MS)

*Note: Based on requirement other methods can be considered for validation

Cleaning validation

Cleaning validation is the validation of the Analytical method used for cleaning the equipment.

7 Guidelines on Analytical Method Validation

The following guidelines are widely considered in the Pharmaceutical industries for Analytical method validation:

  1. The International Conference on Harmonization (ICH)
  2. The United States Environmental Protection Agencies (US EPA)
  3. The American Association of official analytical chemist (AOAC)
  4. The United States Food and Drug Administration (USFDA)
  5. The United States Pharmacopoeia (USP)
  6. The Indian Pharmacopoeia (IP)
  7. ANVISA

7-Step Strategy for the effective Analytical Method Validation

The following 7- Step Strategy should be  followed for any Analytical method validation:

  • Step-1: Evaluate the Analytical Methods: In this step; all requirements and criticality of the method must be evaluated
  • Step-2: Perform pre-validation experiments: Pre-validation is required to avoid any surprises during method validation. This experiment is not part of the validation activities and it is part of development work
  • Step-3: Prepare the validation protocol and in the protocol:
    • Define the validation experiment
    • Define the application, purpose and scope of the method
    • Define the performance parameters and acceptance criteria and
    • Define criteria for re-validation
  • Step-4: Verify relevant characteristics of the equipment
  • Step-5: Qualify/arrange the standards and arrange the samples with valid COA required for the validation
  • Step-6: Perform the Analytical method validation experiment
  • Step-7: Document validation experiments and prepare the method validation report. The method validation report should be approved by all defined persons.

6-Classification of the Chromatographic method

The following are the 6-classification of the chromatographic method:

  • Method versus external standard of the tested analyte (Assay, Content test, Residual solvents and some Impurity profile methods)
  • Methods versus external standards different from the tested analyte (most of the impurity profile methods)
  • Methods of area percentage or Area normalisation method
  • Purity (Solvents, Reagents and some Raw material methods)
  • Impurity profile (for Starting material and Intermediate)
  • Methods of impurities by area per cent or area normalisation for final materials

11 most important Analytical Method Validation Parameters

Analytical method validation parameters
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The following Analytical Validation parameters are performed during method validation:

  1. Specificity
  2. Detection limit (DL)
  3. Quantitation limit (QL)
  4. Precision
  5. Accuracy
  6. Linearity
  7. Range
  8. Relative response factor (RRF)
  9. Robustness
  10. Stability of solutions
  11. Conclusion

Specificity/Selectivity

The ability of an analytical method to measure accurately and specifically the analyte in the presence of components that may be expected to be present in the sample matrix, e.g. impurities, degradation products. Specificity is done by spiking the main substance with all identified available impurities. Impurities which are not mentioned in the monograph should also be considered. PDA detectors and mass spectrometers are used to check peak purity. This test is done in all validation methods.

Acceptance Criteria

  • The peak must be pure
  • Separation between analyte peak and impurities peaks and separation between impurities peak must be observed.

Specificity for Stability Indicating Methods (SIM)

Where a SIM method is validated, the tested material is exposed to stress degradation and stress conditions must include:

  • Heat (solid and solution)
  • Exposure to sunlight (on sun cabinet) (solid and solution)
  • Basic treatment
  • Acidic treatment
  • Oxidation treatment

The assay, peak purity and impurity profile of each treated sample are tested.

Acceptance criteria:

  • The assay should be more than 80%.
  •  Degradation should not be more than 20%.
  • The peak must be pure
  • The impurities separated from the main peak

Procedure and case study : Specificity

Precision

The precision of an analytical procedure expresses the closeness of agreement between a series of
measurements obtained from several samples of the same homogeneous sample under prescribed conditions. Precision is performed for all types of validations. It is subdivided into:

  • System repeatability
  • Method repeatability
  • Intermediate precision
  • Method reproducibility

System Repeatability or system precision

System repeatability is measured by injecting six replicates of standard solutions at nominal concentration.

Acceptance criteria

  • RSD of retention time ≤ 5%
  • For purity test RSD of % area of main analyte ≤ 1%
  • For Related substances or Impurity profile test
    • RSD of % area ≤5% for impurities higher than 0.50%
    • RSD of % area ≤10% for impurities between 0.05% to 0.50%
    • RSD of % area ≤15% for impurities between QL and less than 0.50%
    • RSD of % area will not calculated for impurities lower than quantitation limit

Method Repeatability

The analyte is analysed six times at nominal concentration (six preparation and six injections).

Method reproducibility

In method reproducibility samples are analysed by each analytical and QC analysts using different instruments (make) and different column (lot).

Ideally three samples are taken and analysis is performed in duplicate. If three sample are not available then two sample are taken and one sample is analysed four times and other sample is analyzed in duplicate. If only one sample is available the analysis is performed six times.

Intermediate precision

In In method reproducibility samples are analysed by two different analysts of same labusing different instruments (make) and different column (lot).

Acceptance criteria: Same as Reproducibility test

Procedure and case studies : Precision

Accuracy

Accuracy measures the exactness of the analytical method. It is one of the most critical parameters in
Analytical method validation. It is determined by:

  1. Deviation from linearity: The deviation of the triplicate test of standard solutions with three known concentrations is calculated from the linear regression line
  2. Recovery: In recovery known amount of impurity standard is added to the main substance and recovery is calculated by following the formula👇

Recovery = {(Cs-Cus)⁄Ca}×100

    Where:

  •     Cs= impurity content in the spiked sample (in ppm/percent)
  •     Cus= impurity content in the un-spiked sample (in ppm/percent)
  •     Ca= level of spiking (ppm/percent” added”)

Note: Deviation from linearity and Recovery are performed for drug substances/their stages but for dosage forms, only recovery is performed.

Procedure and case study : Accuracy

Detection Limit (DL)

The lowest concentration of analyte in a sample that can be detected, but not necessarily quantitated as an exact value. It is determined in all types of impurity profile methods including TLC.

The following approaches may be used to calculate DL:

  • Based on visual evaluation: The DL is established by injecting the sample at a minimum concentration where the peak can be reliably detected.
  • Based on Signal-to-Noise ratio: The DL is established by calculating the S/N ratio at minimum concentration. A signal-to-noise ratio  of 3:1 is generally considered to establish the detection limit
  • Based on the Standard deviation of the response and the slope👇

         DL =3.3(σ/S)

Where:

  • σ = the standard deviation of the response    
  • S = the slope of the standard curve

 S may be determined from the calibration curve of the analyte.

The σ may be calculated in the following ways:

  •  Based on the standard deviation of the blank
  •  Based on the calibration curve

Acceptance criteria:

  • Detectability of the peak in three replicates injections. 
  • The S/N ratio should be not less than 3:1

Quantitation Limit (QL)

The lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy under stated experimental conditions.

  • This is required when performing impurity profiles by both the external standard method and the field normalization method.
  • Since TLC methods are the less accurate method. Both DL(detection limit) and QL(quantification limit) have the same value in TLC method.

The following approaches may be used to calculate DL:

  • Based on the Signal-to-Noise ratio
  • Based on the Standard deviation of the response and the slope👇

QL =10(σ/S)

Acceptance criteria:

  • The S/N ratio should be not less than 10:1
  •  RSD limit  depends upon concentration and it should concentration kept as per company SOP

Procedure and case study : DL & QL

Linearity

Linearity is a test that shows the ability to obtain test results that are directly, or by a well-defined
mathematical transformation, proportional to the concentration of analyte in samples within a given range. It is performed on all methods in which standards are used and area per cent methods for final material. A minimum of five standard solutions from two stock solutions are injected once. Standard solutions of at least 50 to 150% of nominal concentration are used for Category I (Assay) analysis.

Acceptance Criteria:

R2 should be less than 0.997 for cat. I analysis and R2 should not be less than 0.990 for Cat. II analysis.

Relative response factor (RRF)

The relative response factor shows the ratio of absorbance between two analytes. It is determined by dividing the slope of specified available impurities by the slope of the reference
substance. Each linearity curve must follow linearity acceptance criteria. It is determined for all available specified ou unspecified impurities of final material.

Procedure and case study: Linearity

Robustness

The measure of the capability of the method to remain unaffected by small deliberate variations in the method parameters during normal usage.

Typical variations are:

  • Influence of variations of pH in a mobile phase
  • Influence in variation in mobile phase composition
  • Columns of different lots or supplier
  • Column temperature
  • Split ratio
  • Incubation temperature
  • Incubation time
  • Whenever the value obtained from a change exceeds acceptance criteria the modification should be repeated with milder change.
  • The Robustness test is performed on all validated methods and the conclusion of the study must be specified in the analytical method

Procedure and Case study: Robustness

Stability of Standard and Sample solution

For assay test

The standard/sample solution is tested for up to 5 days by comparison of the response factor of an old solution (injected as a sample) to freshly prepared standard solutions.

The test for standard solution stability is also valid for sample solution stability because same diluent is used for sample-solution preparations

Acceptance criteria: The test passess the acceptance criteria if % RF difference between freshly prepared standard solution and stability solution is ≤ 2%.

For Related substances test

The sample solution is tested for up to 5 days for related substances stability testing. Result of old samples solution (stability solution) is compared with the freshly prepared sample solutions.

Acceptance criteria: No new peak higher or equal to QL should appear in the old sample solutions

Procedure case study: Stability of solution

Role of Validation Protocol in Analytical Method Validation

Validation protocol is the manual of any Analytical validation work and it is Analytical method specific . It is a pre-approved document that outlines the process for performing analytical method validation. It also includes the purpose, scope and responsibilities of various individuals/departments involved in carrying out the validation.The sensitivity of the method should be considered while designing the protocol to
avoid any surprises or re-work. It is written by the project coordinator and approved by the Analytical Research Manager, Quality Control Manager and Quality Assurance Manager (There may be some changes in the approval process as per the organization’s policy).

The following are the main sections of Validation protocol:

  • Introduction: This section of the analytical validation should include the Standard Test Procedure (STP) Number/Monograph and its corresponding Monograph. It also includes specifications and Analytical validation parameters which will be performed.
  • Methodology: This section includes details about the method which will be validated.
  • Instrument details: This section includes details of all the instruments along with make, model calibration date and calibration due date.
  • Reagent and solvent details: This section includes details of all the instruments along with make, model calibration date and calibration due date.
  • Standards, impurities/impurities marker and sample details: This section includes all the standards, impurities, impurity markers and samples that will be used in the validation.
  • Validation parameters:This section includes procedure details of all of the validation parameters which are going to be performed
  • Precautions:This section includes all the precautions that are required during the validation
  • Conclusion:
  • Abbreviations:This section includes all abbreviations that will be used in the validation

Role of Validation Report in Analytical Method Validation

Once validation work is completed Analytical validation report is prepared based on actual validation work and gets approved by concerned personnel. The following are the important sections of any validation report:

  • Introduction.
  • Methodology
  • Reagent and solvent details
  • Standards, impurities/impurities marker and sample details
  • Analytical Validation parameters
  • Precautions
  • Conclusion:
  • Abbreviations

Work after validation

All limitations observed in robustness testing (such as pH, solvent quality, temperature, buffer concentration etc) during validation should be recorded in the validation report and a as the result monograph should also be updated. If an equivalent column is used in the validation, it will also be updated in the standard test procedure and in the monograph

Applications of AMV report

  • Required by auditors during regulatory audit
  • DMF filing
  • Technology transfer

Conclusion

Different Regulatory Agencies have their own guidelines on analytical method validation whereas different companies have their own guidelines on how to execute Analytical method validation. Therefore the Analytical method validation should be executed in such a way that it meets all the requirements. Hopefully, this article has taken you at the next level and you can now perform analytical validation effectively. You may also want to check out other articles on my blog, such as analytical method mini-validation and Analytical method transfer.

Write your learnings/queries related to this post in the comment section.

Top 25 Interview questions on Analytical method validation

  1. What is Analytical method validation or AMV?
  2. Why Analytical method validation is required?
  3. What are the various guidelines on Analytical method validation?
  4. What are the best strategies for Analytical method validation?
  5. How Analytical method validation parameters are decided?
  6. What should be strategy to perform analytical method validation?
  7. What are the differences between specificity and selectivity?
  8. What are the differences between DL and QL?
  9. What are the different classification of precision?
  10. What are the differences between Accuracy and Recovery?
  11. What are the differences between Accuracy and Recovery?
  12. What is relationship between linearity and range?
  13. How RRF is calculated from linearity?
  14. What are role of DL, QL, precision, specificity, accuracy, recovery, linearity, stability of solution and robustness in analytical method validation?
  15. What are the differences between accuracy and recovery?
  16. How accuracy is related to linearity?
  17. What is the difference between robustness and ruggedness?
  18. How is failure dealt with in the robustness test?
  19. What is the role of Analytical method validation protocol and how it is prepared?
  20. What is the role of Analytical method validation report and how it is prepared?
  21. What are the different classification of the chromatographic method?
  22. What are the different analytical method validation parameters?
  23. What is the RSD limit for analytical method validation?
  24. What are the statistical tools in analytical method validation?
  25. What are the differences between validation and development?
References:

Abbreviations
  • SIM: Stability indicating method
  • HPLC: High pressure liquid chromatography
  • GC: Gas chromatography
  • TLC: Thin Layer chromatography
  • MS: mass spectrometer
  • LC: Liquid chromatography
  • PDA: Photodiode arrays
  • QC: Quality control
  • API: Active pharmaceutical ingredient
  • UV: Ultraviolet
  • FTIR: Fourier transform interferometer
  • AMV: Analytical method validation

Validation

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