Steps for Analytical Methods Validation

Any package of information submitted to foreign regulatory bodies to support new product marketing or clinical trial applications must meet a crucial requirement known as method validation. Analytical procedures, including those published in the pertinent pharmacopeia or other acknowledged standard references, should be verified. All test procedures should have their appropriateness well documented and confirmed under their intended application.

Accuracy, precision (repeatability and intermediate precision), specificity, detection limit, quantitation limit, linearity, range, and robustness are the most often used typical validation criteria for different types of tests (Table 1). The stability of analytical results and system suitability should also be included in the information on technique validation.

Table-1: ICH, USP, and FDA Methods Validation Characteristics Requirements for Various Types of Tests

Validation Characteristics	Assay	Testing for Impurities		Identification
		Quantitative	Limit
Accuracy	Yes	Yes	No	No
Precision - Repeatability	Yes	Yes	No	No
Precision - Intermediate Precision	Yes1	Yes*	No	No
Specificity	Yes	Yes	Yes	Yes
Detection limit	No	No	Yes	No
Quantitation limit	No	Yes	No	No
Linearity	Yes	Yes	No	No
Range	Yes	Yes	No	No
Robustness	Yes	Yes	No	No

Prepare a Protocol

The first stage in method validation is to create a protocol, preferably one that is documented, has clear, step-by-step instructions, and has been validated before being used. This paper discusses this strategy. The proposed acceptance criteria may be altered depending on the technique employed, the required accuracy, and the required sensitivity. (Note: The characterization research forms the basis for the majority of the acceptance criteria.) Additionally, depending on sound scientific judgment, some tests may be skipped, and the number of duplicates may be decreased or increased.

A test method is considered validated when it meets the acceptance criteria of a validation protocol. This paper is a step-by-step practical guide for preparing protocols and performing test methods validation with reference to High-Performance Liquid Chromatography (HPLC) (use similar criteria for all other instrumental test method validation) in the quality system compliance industry.

Prepare Analytical Method Validation Protocol as per the format given in the below link.

Analytical Method Validation Protocol

Procedure for Analytical Performance Characteristics

The analytical system must be properly built, maintained, calibrated, and verified before beginning the task of methods validation. All employees who will carry out the validation tests must have received the necessary training. The CFT must approve the method validation methodology before it may be used.

Specificity

Test procedure

The specificity of the assay method will be investigated by injecting of the extracted placebo to demonstrate the absence of interference with the elution of the analyte.

Documentation

Print chromatograms.

Acceptance criteria

The excipient compounds must not interfere with the analysis of the targeted analyte.

Linearity

Test procedure

Standard solutions will be prepared at six concentrations, typically 25, 50, 75, 100, 150, and 200% of the target concentration. Three individually prepared replicates at each concentration will be analyzed. The method of standard preparation and the number of injections will be the same as used in the final procedure.

Documentation

Record results on a data sheet. Calculate the mean, standard deviation, and Relative Standard Deviation (RSD) for each concentration. Plot concentration (x-axis) versus mean response (y-axis) for each concentration. Calculate the regression equation and coefficient of determination (r2). Record these calculations on the datasheet.

ALSO READ: Short Information on Cleaning Validation

Acceptance criteria

The correlation coefficient for six concentration levels will be ≥ 0.999 for the range of 80 to 120% of the target concentration. The y-intercept must be ≤ 2% of the target concentration-response. A plot of response factor versus concentration must show all values within 2.5% of the target level response factor, for concentrations between 80 and 120% of the target concentration.

Concentration (mg/ml)	Concentration as % of Analyte Target	Peak Area (mean of 3 Injections)	Peak Area RSD (%)
5	25
10	50
15	75
20	100
30	150
40	200
The equation for regression line =		Correlation coefficient (r2) =

Range

Test procedure

The data obtained during the linearity and accuracy studies will be used to assess the range of the method.

The precision data used for this assessment is the precision of the three replicate samples analyzed at each level in the accuracy studies.

Documentation

Record the range on the datasheet.

Acceptance criteria

The acceptable range will be defined as the concentration interval over which linearity and accuracy are obtained per the above criteria, and in addition, that yields a precision of ≤ 3% RSD.

Accuracy

Test procedure

Spiked samples will be prepared at three concentrations over the range of 50 to 150% of the target concentration.

Three individually prepared replicates at each concentration will be analyzed. When it is impossible or difficult to prepare known placebos, use a low concentration of a known standard.

Documentation

For each sample, report the theoretical value, assay value, and percent recovery. Calculate the mean, standard deviation, RSD, and percent recovery for all samples. Record results on the datasheet.

Acceptance criteria

The mean recovery will be within 90 to 110% of the theoretical value for non-regulated products. For the U.S. pharmaceutical industry, 100 ± 2% is typical for an assay of an active ingredient in a drug product over the range of 80 to 120% of the target concentration. Lower percent recoveries may be acceptable based on the needs of the methods.

Sample	Percent of Nominal (mean of three injections)	Amount of Standard (mg)		Recovery (%)
		Spiked	Found
1	75
2	100
3	150
Mean
SD
RSD%

Precision

Repeatability

Test procedure

One sample solution containing the target level of the analyte will be prepared. Ten replicates will be made from this sample solution according to the final method procedure. Record the retention time, peak area, and peak height on the datasheet. Calculate the mean, standard deviation, and RSD.

Acceptance criteria

The typical RSD should be 1% for drug substances and drug products and ± 2% for bulk drugs and finished products. 

Injection No.	Retention Time (min)	Peak Area	Peak Height
Replicate 1
Replicate 2
Replicate 3
Replicate 4
Replicate 5
Replicate 6
Replicate 7
Replicate 8
Replicate 9
Replicate 10
Mean
SD
RSD%

ALSO READ: Process Validation Protocol for Oral Rehydration Salt

Intermediate Precision

Test procedure

Intermediate precision (within-laboratory variation) will be demonstrated by two analysts, using two HPLC systems on different days and evaluating the relative percent purity data across the two HPLC systems at three

concentration levels (50%, 100%, 150%) that cover the analyte assay method range 80 to 120%.

Documentation

Record the relative % purity (% area) of each concentration on the datasheet.

Calculate the operators' and instruments' mean, standard deviation, and RSD.

Acceptance criteria

The assay results obtained by two operators using two instruments on different days should have a statistical RSD ≤ 2%.

	Relative % Purity (% area)
	Instrument 1			Instrument 2
Sample	S1 (50%)	S2 (100%)	S3 (150%)	S1 (50%)	S2 (100%)	S3 (150%)
Operator 1, Day 1
Operator 1, Day 2
Operator 2, Day 1
Operator 2, Day 2
Mean (Instrument)
Mean (Operators)
RSD%	S1 + S1	S2 + S2	S3 + S3
Instruments
Operators

Limit of Detection

Test procedure

The lowest concentration of the standard solution will be determined by sequentially diluting the sample. Six replicates will be made from this sample solution.

Documentation

Print the chromatogram and record the lowest detectable concentration and RSD on the datasheet.

Acceptance criteria

The ICH references a signal-to-noise ratio of 3:1. Some analysts calculate the standard deviation of the signal (or response) of a number of blank samples and then multiply this number by two to estimate the signal at the limit of detection.

Record sample data results: (e.g., concentration, S/N ratio, RSD%)

Limit of Quantitation

Test procedure

Establish the lowest concentration at which an analyte in the sample matrix can be determined with the accuracy and precision required for the method in question. This value may be the lowest concentration in the standard curve. Make six replicates from this solution.

Documentation

Print the chromatogram and record the lowest quantified concentration and RSD on the datasheet. Provide data that demonstrate the accuracy and precision required in the acceptance criteria.

ALSO READ: SOP for Transport Validation

Acceptance criteria

The limit of quantitation for chromatographic methods has been described as the concentration that gives a signal-to-noise ratio (a peak with height at least ten times as high as the baseline noise level) of 10:1.

Record sample data results: (e.g., concentration, S/N ratio, RSD%)

System Suitability

Test procedure

System suitability tests will be performed on both HPLC systems to determine the accuracy and precision of the system by injecting six injections of a solution containing analyte at 100% of test concentration. The following parameters will be determined: plate count, tailing factors, resolution, and reproducibility (% RSD of retention time, peak area, and height for six injections).

Documentation

Print the chromatogram and record the data on the datasheet

Acceptance criteria

Retention factor (k): the peak of interest should be well resolved from other peaks and the void volume; generally k should be ≥ 2.0. 

Resolution (Rs): Rs should be ≥ 2 between the peak of interest and the closest eluted peak, which is potentially interfering (impurity, excipient, and degradation product). 

Reproducibility: RSD for peak area, height, and retention time will be 1% for six injections.

Tailing factor (T): T should be 2. 

Theoretical plates (N): ≥ 2000.

System Suitability Parameter	Acceptance Criteria	Results		Criteria Met/ Not Met
		HPLC 1	HPLC 2
Injection precision for retention time (min)	RSD ≤ 1%
Injection precision for peak area (n = 6)	RSD ≤ 1%
Injection precision for peak height	RSD ≤ 1%
Resolution (Rs)	Rs = ≥ 2.0
USP tailing factor (T)	T = ≤ 2.0
Capacity factor (k)	K = ≥ 2.0
Theoretical plates (N)	N = ≥ 2000

Robustness

Robustness measures the capacity of an analytical method to remain unaffected by small but deliberate variations in method parameters.

Robustness provides some indication of the reliability of an analytical method during normal usage. Parameters, which will be investigated, are the percent organic content in the mobile phase or gradient ramp, pH of the mobile phase, buffer concentration, temperature, and injection volume. These parameters may be evaluated one factor at a time or simultaneously as part of a factorial experiment.

The chromatography obtained for a sample containing representative impurities, when using a modified parameter(s), will be compared to the chromatography obtained using the target parameters. The effects of the following changes in chromatographic conditions will be determined:

methanol content in the mobile phase adjusted by ±2%, mobile phase pH adjusted by ± 0.1 pH units, column temperature adjusted by ± 5°C. If these changes are within the limits that produce acceptable chromatography, they will be incorporated into the method procedure.

ALSO READ: Analytical Method Validation Protocol for Titration Method