Validation of Analytical

Method
Ashok Suthar Regulatory Associate Regulatory Affairs

Amneal Pharmaceuticals

Definition: Method validation is the process of proving that an analytical method is acceptable for its intended purposes. METHOD VALIDATION = ERROR ASSESSMENT

Why Method Validation is Important?

1. Develops confidence in using the method & Proof that Method is suitable for its intended purpose, The purpose of analytical measurement is to get consistent, reliable and accurate data.

Incorrect measurement results can lead to tremendous costs.

2. Regulatory requirement, Equal importance for those working in a regulated and in an accredited environment. U.S. FDA, ISO etc.

When to be validated?
Partial validation after development of method. Complete validation after manufacturing formula is finalized.

Which methods are to be validated

Compendial: Pharmacopoeial method

Verification of suitability of method

Non compendial methods: Laboratory developed methods. Pharmacopoeial methods used outside its scope.

Validation: Prior Considerations

Suitability of Instrument
Status of Qualification and Calibration

Suitability of Materials
Status of Reference Standards, Reagents, Placebo Lots

Suitability of Analyst
Status of Training and Qualification Records

Suitability of Documentation
Written and approved standard test procedure and proper approved protocol with pre-established acceptance criteria

Validation Activity Including the Complete Analytical Procedure

Sampling

Sample Preparation

Analysis

Data Evaluation Reporting

Validation Step

Define the application, purpose and scope of the

method. Analytes? Concentration?

Develop a analytical method.

Develop a validation protocol. Qualification of instrument. Qualify/train operator

Validation Step

Qualification of material. Perform pre-validation experiments. Adjust method parameters and/or acceptance criteria if necessary. Perform full validation experiments.

Develop Procedures for executing the method in routine analysis.

Document validation experiments and results in the validation report.

Verification vs. Validation

Compendial vs. Non-compendial Methods

Compendial methods-Verification
Regulatory analytical procedure in USP/NF Non- compendial methods-Validation Alternative analytical procedure proposed by the applicant for use instead of the regulatory analytical procedure

BACKGROUND-LAB METHOD FLOW

Method Development

Method Validation

Method Transfer

Approved

ICH/USP Validation Requirements

Precision
Repeatability

Specificity

Intermediate Precision Reproducibility Limit of Detection Limit of Quantitation Robustness

Linearity
Range Accuracy

System suitability

Validation Parameters

Assay / CU

Dissolution

Impurities

Specificity Linearity and Range Accuracy Precision Robustness

Specificity Specificity Linearity and Range LOD & LOQ Accuracy Linearity and Range Precision Accuracy Robustness Precision Robustness

Specificity

Ability of an analytical method

to measure the
analyte free from interference due to Selectivity Bias

other components.

Specificity: ICH/USP
The ability to measure accurately and
specifically the analyte in the presence of components that may be expected to be

present in the matrix

The degree of interference Active Ingredients

Excipients
Impurities (synthetic precursors, enantiomers)

Degradation Products
Placebo Ingredients

Combination of 2 or more analytical procedures may be

required to achieve necessary level of discrimination (i.e. LC-MS, etc.)

Stability indicating analytical methods should always be specific

Analysts should ascertain whether the peaks within a

sample chromatogram are pure or consist of more than one compound. Therefore should know how many compounds are in the sample or use procedures to detect peak purity

Linearity

Ability of an assay to elicit a direct and

proportional
response to changes in analyte concentration.

Contd.
The Ability of the method to obtain test results that are directly proportional to concentration within a given range

Method: dilution of stock solution/separate

weightings Expressed as the variance of the slope of the

regression line
Correlation coefficient, y-intercept, slope of regression line and residual sum of squares

should be presented together with plot of the

data

Range
The interval between the
upper and lower concentrations of analyte

in the sample that have

been demonstrate to have a suitable level of precision, accuracy, and linearity.

Range

Normally derived from Linearity studies. Established by confirming that the method provides

acceptable degree of linearity, accuracy, and

precision. Specific range dependent upon intended application of the procedure.

Accuracy

Closeness of the test results obtained by the method to the true value.

Precision
Ball Ball Ball Ball Ball Ball Ball Ball Strike Strike Strike Strike Strike Strike

The closeness of agreement

(degree of scatter) between a

series of measurements obtained from multiple samplings of the same homogeneous sample. Should be investigated using homogeneous, authentic samples.

Accuracy Vs Precision

Inaccurate & imprecise

Inaccurate but precise

Accurate but imprecise

Precision Considered at 3 Levels

Repeatability Intermediate Precision Reproducibility

Repeatability

Express the precision under the same operating conditions over a short interval of time.

Also referred to as Intra-assay precision

Intermediate Precision
Express within-laboratory variations. Expressed in terms of standard deviation, relative standard deviation (coefficient of variation) and confidence interval. Studies should include varying days, analysts,

Depends on the
circumstances under which the procedure is intended

to be used.

Known as part of Ruggedness in

USP (Different Analysts, Different Laboratories, Different Instruments,

equipment, etc.

Reproducibility

Definition: Ability to reproduce data within the predefined precision

Determination: SD, RSD and confidence interval

Repeatability test at two different labs.

Reproducibility Study
Lab 1
Day 1 Day 2

Lab 2
Day 1 Day 2

Lab 3
Day 1 Day 2

Analyst 1 3 Preps

Analyst 2 3 Preps

Analyst 1 3 Preps

Analyst 2 3 Preps

Analyst 1 3 Preps

Analyst 2 3 Preps

Detection Limit (DL)

Quantitation Limit (QL)

Lowest amount of analyte in a

sample that can be detected but not necessarily quantitated. Estimated by Signal to Noise Ratio of 3:1.

Lowest amount of analyte in a

sample that can be quantified with suitable accuracy and precision. Estimated by Signal to Noise Ratio of 10:1.

LOD, LOQ and SNR

Limit of Quantitation (LOQ)
Limit of Detection (LOD) Signal to Noise Ratio (SNR)
Peak B LOQ

Peak A LOD Baseline noise

Robustness

Definition: Capacity to remain unaffected by

small but deliberate variations in method

parameters

Determination:

Comparison

results

under

differing conditions with precision under normal conditions Variations may include: stability of analytical solution, variation of pH in a mobile phase, different column (lot/supplier), temperature,

Stability of analytical solution

Solutes may readily decompose prior to chromatographic investigations e.g. during sample preparation, extraction, cleanup, phase transfer or storage of prepared vials (refrigerators or automatic sampler). Method development should investigate the stability of the Analytes AND standards.
System stability Stability of the samples being analyzed in a sample solutio n. e.g. 1 48 hours using a single solution. should be determined by replicate analysis of the sample solution.

SYSTEM SUITABILITY

The checking of a system, before or during analysis of unknowns, to ensure system performance.

No sample analysis is acceptable unless the requirements f or system suitability have been met. (USP Chapter 621)
Plate Count, Tailing, Resolution Determination of reproducibility (%RSD) For %RSD < 2.0%, Five replicates For %RSD > 2.0%, Six replicates System Suitability "Sample - A mixture of main components and expected by-products utilized to determine system suitability

Whenever There is a Significant change in Equipment or Reagents System Suitability Testing Should be Performed (USP Chapter 621)

Confuse of Precision Terms

Repeatability Intermediate Precision

Reproducibility Ruggedness Robustness

Precision Terms

Instrument Precision
Repeatability Intermediate Precision Reproducibility Ruggedness Robustness

- 6 Standard Injections
- One Analysis (6 preps) - Two Analyses - Two different Lab. - Many Variables - Intentional Changes

Robustness Variations

All Assays

-Sample Prep Manipulation -Extraction Time -Mobile Phase Composition -Different Columns -Temperature -Flow Rate

HPLC Assays

GC Assays

-Different Columns -Temperature

-Flow Rate

Revalidation
Change in the analytical procedure, drug substance, drug product, the changes, may necessitate revalidation of the analytical procedures.

The degree of revalidation depends on the nature of the change.

FDA intends to provide guidance in the future on post-approval changes in analytical procedures.
Revalidation should accompany formulation changes (new samples with new compounds or new matric es) manufacturing batch changes new analysts with different skills, new instruments with different characteristics, new location with different environmental conditions, new chemicals and/or reference standards and modification of analytical parameters.

Validation Report
Objective and scope of the method (applicability, type). Summary of methodology. Type of compounds and matrix. All chemicals, reagents, reference standards, QC samples with purity, grade, their s ource or detailed instructions on their preparation. Procedures for quality checks of standards and chemicals used. Method parameters. Critical parameters taken from robustness testing. Listing of equipment and its functional and performance requirements, e.g., cell di mensions, baseline noise and column temperature range. Detailed conditions on conduct of experiments, including sample preparation Statistical procedures and representative calculations. Procedures for QC in routine analyses, e.g., system suitability tests. Representative plots, e.g., chromatograms, spectra and calibration curves. Method acceptance limit performance data and expected uncertainty of measurem ent results. Criteria for revalidation. The person's) who developed and validated the method. References (if any). Summary and conclusions.

General requirements
Qualified and calibrated instruments
Documented methods Reliable reference standards

Qualified analysts
Sample integrity Change control (e.g., synthesis, FPP composition) Analytical methods should be used within GMP and GLP environments, and must be developed using the protocols and acceptance criteria set out in the ICH guidelines Q2 (R1)

Thank you