Checklist for Analytical Method Validation

Analytical Method Validation is to be performed for new analysis methods or for current methods when any changes are made to the procedure, the composition of the drug product, and the synthesis of the drug substances. Common types of analytical procedures that can be validated.

What is meant by method validation?

According to the FDA, “Analytical method validation is the process of demonstrating that an analytical procedure is suitable for its intended purpose. The methodology and objective of the analytical procedures should be clearly defined and understood before initiating validation studies.

What is the purpose of method validation?

Method validation data provide information that enables the comparability of results from samples analyzed in different laboratories and using different methods to be assessed. Method validation is an essential part of the process of ensuring that measurement results reported to customers are correct.

What is method validation in the laboratory?

Method validation is a demonstration of the method's suitability by determining the accuracy of the test results as well as the uncertainty and traceability of measurements. Method validation is needed for proving whether the new method is fit for the purpose or specified samples.

What are the types of validation?

Prospective validation

Retrospective validation.

Concurrent validation.

Revalidation.

 

What are LOD and LOQ in method validation?

The limit of detection (LOD) and limit of quantification (LOQ) are two important performance characteristics in method validation. LOD and LOQ are terms used to describe the smallest concentration of an analyte that can be reliably measured by an analytical procedure.

What is the difference between method validation and verification?

In conclusion, method validation is usually applied to an “in-house method” developed by a laboratory; while method verification is applied to a “compendia method or previously validated method” when it is being used in a particular laboratory for the first time.

What is the difference between method validation and Process Validation?

The major difference in the process and product validation is their methodologies. Process validation is aimed at ensuring different inputs will guarantee a similar end product while product validation aims to ensure the end product works effectively to its intended purpose; meeting user needs and requirements.

What are the parameters of validation?

The Validation parameters are accuracy, precision, linearity and application range, the limit of detection (LOD), the limit of quantitation (LOQ), selectivity/specificity, recovery, and robustness/ruggedness.

Why is analytical method validation important?

Validation of analytical test methods is important for every stage of the drug development life cycle. Methods must be shown to be acceptable for their intended use and must provide accurate and reliable results at every stage of development, from pre-clinical trials through approved and marketed products.

ALSO READ: Difference between Qualification and Validation

How do you validate a test method?

The accuracy of an analytical procedure is defined as how close the test results of the parameters are for a specific analyte compared to the true measure of these parameters.

1. Precision.
2. Specificity.
3. Limit of detection.
4. Limit of quantitation.
5. Linearity.
6. Range.
7. Robustness.

How is the LOD value calculated?

LOD may also be calculated based on the standard deviation of the response (Sy) of the curve and the slope of the calibration curve (S) at levels approximating the LOD according to the formula: LOD = 3.3(Sy/S).

What is the blank limit?

The limit of blank (also known as the critical value) is the highest quantity value that is likely to be observed, with a stated probability, for a blank material. Related concepts. Limit of detection (LOD) Related tasks. Estimating the detection limit of a measurement system.

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What is the limit of linearity?

Limit of linearity (LOL). The concentration at which the calibration curve departs from linearity. The extension from the LOQ to LOL is called the dynamic range. A well-defined relationship between detector response and analyte concentration is crucial for quantitative analysis.

Checklist for Analytical Method Validation (Chemical)

Assay/Related Substances

TEST	ASSAY/RELATED SUBSTANCES
PARAMETER	No.	DOCUMENTS REQUIRED	AVAILABILITY
Specificity	1	Testing Method
	2	Acceptance criteria
	3	Chromatogram/spectrum for the following solutions:
		Standard
		Sample
		Blank/Placebo
		Spike solution
		System suitability tests
	4	Impurities available
		Peak purity for PDA detector or spike solution for non-PDA detector
		Relative Retention Time (RRT)
		Resolution (R) where applicable
	5	Impurities not available
		Stress data (minimum data for humidity, heat and light)
		Peak purity
		Relative Retention Time (RRT)
		Resolution (R) where applicable
Linearity	1	Testing Method
	2	Acceptance criteria
	3	Minimum five (5) levels of standard solutions
	4	Data for linear regression equation, Y-intercept, slope, r2 and linearity graph

Range	1	For the assay of drug substances: 80% - 120% of working concentration (WC)
	2	For the determination of an impurity: from the reporting level of an impurity to 120% of the specification
Accuracy	1	Testing Method
	2	Acceptance criteria
	3	Minimum three (3) levels of concentration in triplicates covering the specified range
	4	Result: reported as percent recovery by the assay of known added amount of analyte in the sample, OR as the difference between the mean and the accepted true value together with the confidence intervals
Precision (Repeatability)	1	Testing Method (using sample/product as the test solution)
	2	Acceptance criteria
	3	Minimum three (3) levels of concentration in triplicates covering the specified range, OR minimum six (6) replicates at 100% of the WC
	4	Result: SD, RSD and confidence Interval
Precision (intermediate precision/ ruggedness)	1	Testing Method (using sample/product as the test solution)
	2	Acceptance criteria
	3	Minimum three (3) levels of concentration in triplicates covering the specified range, OR minimum six (6) replicates at 100% of the WC
	4	Cover at least 2 parameters among variation of analyst, date and equipment
	5	Result: SD, RSD and confidence Interval

ALSO READ: Steps for Analytical Methods Validation

Detection Limit	1	Testing Method: visual observation/signal-to-noise / standard deviation of the response and the slope
	2	If based on standard deviation of the response and the slope method
		Minimum five (5) levels of standard solutions
		Peak area values for all concentrations
		Data for linear regression equation, Y-intercept, slope, r2 and linearity graph.
	3	Calculation/formulation where applicable
	4	Related Chromatogram(s) at LOD
	5	Value of detection limit
Quantitation Limit	1	Testing Method: visual observation/signal-to-noise / standard deviation of the response and the slope
	2	if based on the visual observation method, accuracy and precision data at LOQ must be provided
	3	If based on the calibration curve method
		Minimum five (5) levels of standard solutions
		Peak area values for all concentrations
		Data for linear regression equation, Y-intercept, slope, r2 and linearity graph.
	5	Calculation/formulation where applicable
	6	Value of quantitation limit
System Suitability Testing	1	RSD, tailing factor, theoretical plate
	2	Resolution (if two or more components)
Robustness (not mandatory)	1	Testing Method
	2	Acceptance criteria
	3	Result: refer to acceptance criteria for accuracy and precision (repeatability)

Checklist for Analytical Method Validation (Chemical)

Dissolution

TEST	DISSOLUTION
PARAMETER	No.	DOCUMENTS REQUIRED	AVAILABILITY
Specificity	1	Testing Method
	2	Acceptance criteria
	3	Chromatogram/spectrum for the following solutions:
		Standard
		Sample
		Blank/Placebo
		Spike solution
		System suitability tests
Linearity	1	Testing Method
	2	Acceptance criteria
	3	Minimum five (5) levels of standard solutions
	4	Data for linear regression equation, Y-intercept, slope, r2, and linearity graph.
Range	1	Dissolution testing: ± 20% over the specified range Example 1: if the specification is NLT 75% (Q) of the labeled amount is dissolved in 45 minutes, the validated range would be 60 – 100% of the label claim Example 2: if the specification for a controlled released product covers a region from 20% after 1 hour, up to 90%, after 24 hours, the validated range would be 0 – 110% of the label claim
Accuracy	1	Testing Method
	2	Acceptance criteria
	3	Minimum three (3) levels of concentration in triplicates covering the specified range
	4	Result: reported as percent recovery by the assay of known added amount of analyte in the sample, OR as the difference between the mean and the accepted true value together with the confidence intervals

ALSO READ: Analytical Method Validation Protocol for Nystatin

Precision (Repeatability)	1	Testing Method (using sample/product as the test solution)
	2	Acceptance criteria
	3	Minimum three (3) levels of concentration in triplicates covering the specified range, OR minimum six (6) replicates at 100% of the WC
	4	Result: SD, RSD and confidence Interval
Precision (intermediate precision/ ruggedness)	1	Testing Method (using sample/product as the test solution)
	2	Acceptance criteria
	3	Minimum three (3) levels of concentration in triplicates covering the specified range, OR minimum six (6) replicates at 100% of the WC
	4	Cover at least 2 parameters among variations of analyst, date and equipment
	5	Result: SD, RSD and confidence Interval
System Suitability Testing	1	RSD, tailing factor, theoretical plate
	2	Resolution (if two or more components)
Robustness (Not Mandatory)	1	Testing Method
	2	Acceptance criteria
	3	Result: refer to acceptance criteria for accuracy and precision (repeatability)

Commonly Acceptance Criteria

PARAMETER	ACCEPTANCE CRITERIA
Specificity	Absence of interfering peaks in the placebo, impurity demonstrates specificity
	Pass peak purity test (particularly for related substances test)
Linearity	r2 ≥ 0.995
	y-intercept at 100% working concentration ≤ 2%
Accuracy	Measured recovery within 95% - 105% or mean difference ± 2% & CI
Precision (Repeatability)	RSD ≤ 2.0% & CI
Precision (intermediate precision/ ruggedness)	RSD ≤ 2.0% & CI or mean difference ± 2% & CI
Detection Limit	LOD peak must be visible
	If based on a standard deviation of the response and the slope method, DL = 3.3 Ó/S
	If based on signal to noise, S/N= 3:1 or 2:1
Quantitation Limit	if based on the visual observation method, accuracy and precision data at LOQ must be ± 20%
	If based on a standard deviation of the response and the slope method, DL = 10 Ó/S
	If based on signal to noise, S/N= 3:1 or 2:1
System Suitability Testing	RSD ≤ 2%
	Theoretical plate/column efficiency, N ≥ 2000
	Tailing factor < 2
	Resolution > 2
Robustness (Not mandatory)	Refer to acceptance criteria for accuracy and precision (repeatability)

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