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Analytical Method Validation

An Analytical Procedure is the most important key in Analytical Method Validation. The analytical procedure defines characteristics of Drug Product or Drug Substance also gives acceptance criteria for the same. there are two Types of Analytical Procedures first is Specifications and standard test method in Pharmacopoeias or Pharmacopoeial methods and second one Non-Pharmacopoeial method or method which is developed In-house and approved by the National Regulatory Authority.

What Are the types of Analytical Method Validation to be Validated..??

Analytical method validation is mainly performed for below-mentioned test procedures;

  1. Identification tests;
  2. Quantitative tests for Impurities content;
  3. Limit tests for the control of Impurities;
  4. Quantitative tests of the active moiety in samples of the drug substance or drug product or other selected component(s) in the drug product.

Analytical method validation characteristics which should be considered during performing of method validation;

  1. Accuracy.
  2. Precision.
  3. Specificity.
  4. Detection Limit.
  5. Quantification Limit.
  6. Linearity.
  7. Range.

Characteristics to consider during Analytical Method Validation;( ICH Q2)

Type of analytical procedure characteristics IDENTIFICATION TESTING FOR IMPURITIES ASSAY

– dissolution (measurement only)

– content/potency

Quantitative limit
Accuracy + +
Precision

– Repeatability

– Interm. Precision

+

+ (1)

+

+(1)

Specificity (2) + + + +
Detection Limit -(3) +
Quantitation Limit +
Linearity + +
Range + +

where;

  • signifies that this characteristic is not normally evaluated
  • signifies that this characteristic is normally evaluated

(1) In cases where reproducibility (see glossary) has been performed, intermediate precision is not needed

(2) Lack of specificity of one analytical procedure could be compensated by other supporting analytical procedure(s)

(3) Maybe needed in some cases

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 an accepted reference value and the value found. This is sometimes termed trueness.  It is normally established on samples of the material to be examined that have been prepared for quantitative accuracy. Accuracy should be established across the specified range of the analytical procedure.

Assay-

Drug Substance
Several methods of determining accuracy are available:
a) application of an analytical procedure to an analyte of known purity (e.g. reference material);
b) comparison of the results of the proposed analytical procedure with those of a second well-characterized procedure, the accuracy of which is stated and/or defined (independent procedure, see 1.2.);
c) accuracy may be inferred once precision, linearity, and specificity have been established.

Drug Product
Several methods for determining accuracy are available:
a) application of the analytical procedure to synthetic mixtures of the drug product components to which known quantities of the drug substance to be analyzed have been added;
b) in cases where it is impossible to obtain samples of all drug product components , it may be acceptable either to add known quantities of the analyte to the drug product or to compare the results obtained from a second, well characterized procedure, the accuracy of which is stated and/or defined (independent procedure, see 1.2.);
c) accuracy may be inferred once precision, linearity, and specificity have been established.

Impurities (Quantitation):-
Accuracy should be assessed on samples (drug substance/drug product) spiked with known amounts of impurities.
In cases where it is impossible to obtain samples of certain impurities and/or degradation products, it is considered acceptable to compare results obtained by an independent procedure (see 1.2.). The response factor of the drug substance can be used. It should be clear how the individual or total impurities are to be determined e.g., weight/weight or area percent, in all cases with respect to the major analyte.

Acceptance Criteria:-

This test is mainly to check the recovery of API with Placebo. analyst have to prepare sample solution in triplicate of API with Placebo at different minimum 3 concentrations (e.g. 80%, 100% & 120%) it will give nine results.

acceptance criteria of this test should %recovery at each concentration  ±5 % and % RSD should be not more than 5.0. Accuracy should be reported as percent recovery by the assay of the 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:-

System Precision: –

Standard solutions should be prepared as per method  a minimum of 6 determinations at 100% of the test concentration or  a minimum of 9 determinations covering the specified range for the procedure (e.g., 3 concentrations/3 replicates each); 

Intermediate Precision:-  

The extent to which intermediate precision should be established depends on the circumstances under which the procedure is intended to be used. The applicant should establish the effects of random events on the precision of the analytical procedure. Typical variations to be studied include days, analysts, equipment, etc. It is not considered necessary to study these effects individually. The use of experimental design (matrix) is encouraged.

Perform ruggedness with a different analyst on a different day by calculating % RSD

Method Precision:- 

Method Precision to reproduce results through the same method with six samples

SPECIFICITY:-
An investigation of specificity should be conducted during the validation of identification tests, the determination of impurities and the assay. The procedures used to demonstrate specificity will depend on the intended objective of the analytical procedure. It is not always possible to demonstrate that an analytical procedure is specific for a particular analyte (complete discrimination). In this case, a combination of two or more analytical procedures is recommended to achieve the necessary level of discrimination.

Identification:-
Suitable identification tests should be able to discriminate between compounds of closely related structures which are likely to be present. The discrimination of a procedure may be confirmed by obtaining positive results (perhaps by comparison with a known reference material) from samples containing the analyte, coupled with negative results from samples which do not contain the analyte. In addition, the identification test may be applied to materials structurally similar to or closely related to the analyte to confirm that a positive response is not obtained. The choice of such potentially interfering materials should be based on sound scientific judgment with a consideration of the interferences that could occur.

Assay and Impurity Test(s):-
For chromatographic procedures, representative chromatograms should be used to demonstrate specificity and individual components should be appropriately labeled. Similar considerations should be given to other separation techniques. Critical separations in chromatography should be investigated at an appropriate level. For critical separations, specificity can be demonstrated by the resolution of the two components which elute closest to each other.

In cases where a non-specific assay is used, other supporting analytical procedures should be used to demonstrate overall specificity. For example, where titration is adopted to assay the drug substance for release, the combination of the assay and a suitable test for impurities can be used.

The approach is similar for both assay and impurity tests: Impurities are available For the assay , this should involve demonstration of the discrimination of the analyte in the presence of impurities and/or excipients; practically, this can be done by spiking pure substances (drug substance or drug product) with appropriate levels of impurities and/or excipients and demonstrating that the assay result is unaffected by the presence of these materials (by comparison with the assay result obtained on unspiked samples). For the impurity test, the discrimination may be established by spiking drug substance or drug product with appropriate levels of impurities and demonstrating the separation of these impurities individually and/or from other components in the sample matrix.
Impurities are not available:-
If impurity or degradation product standards are unavailable, specificity may be demonstrated by comparing the test results of samples containing impurities or degradation products to a second well-characterized procedure e.g.: pharmacopoeial method or other validated analytical procedure (independent procedure). As appropriate, this should include samples stored under relevant stress conditions: light, heat, humidity, acid/base hydrolysis, and oxidation.

  • for the assay, the two results should be compared;
  • for the impurity tests, the impurity profiles should be compared. Peak purity tests may be useful to show that the analyte chromatographic peak is not attributable to more than one component (e.g., diode array, mass spectrometry).

DETECTION LIMIT:- 
Several approaches for determining the detection limit are possible, depending on whether the procedure is a non-instrumental or instrumental. Approaches other than those listed below may be acceptable.
Based on Visual Evaluation-
The visual evaluation may be used for non-instrumental methods but may also be used with instrumental methods.
The detection limit is determined by the analysis of samples with known concentrations of analyte and by establishing the minimum level at which the analyte can be reliably detected.
Based on Signal-to-Noise-
This approach can only be applied to analytical procedures which exhibit baseline noise. Determination of the signal-to-noise ratio is performed by comparing measured signals from samples with known low concentrations of analyte with those of blank samples and establishing the minimum concentration at which the analyte can be reliably detected.

A signal-to-noise ratio between 3 or 2:1 is generally considered acceptable for estimating the detection limit.

Based on the Standard Deviation of the Response and the Slope The detection limit (DL) may be expressed as:
DL =    3.3 σ  
S
where,

σ = the standard deviation of the response
S = the slope of the calibration curve
The slope S may be estimated from the calibration curve of the analyte. The estimate of σ may be carried out in a variety of ways, for example: Based on the Standard Deviation of the Blank Measurement of the magnitude of analytical background response is performed by analyzing an appropriate number of blank samples and calculating the standard deviation of these responses.
Based on the Calibration Curve:-
A specific calibration curve should be studied using samples containing an analyte in the range of DL. The residual standard deviation of a regression line or the standard deviation of y-intercepts of regression lines may be used as the standard deviation.

Recommended Data – The detection limit and the method used for determining the detection limit should be presented. If DL is determined based on visual evaluation or based on the signal to noise ratio, the presentation of the relevant chromatograms is considered acceptable for justification.

In cases where an estimated value for the detection limit is obtained by calculation or extrapolation, this estimate may subsequently be validated by the independent analysis of a suitable number of samples known to be near or prepared at the detection limit.

QUANTITATION LIMIT:-
Several approaches for determining the quantitation limit are possible, depending on whether the procedure is a non-instrumental or instrumental. Approaches other than those listed below may be acceptable.
Based on Visual Evaluation:-
A visual evaluation may be used for non-instrumental methods but may also be used with instrumental methods. The quantitation limit is generally determined by the analysis of samples with known concentrations of analyte and by establishing the minimum level at which the analyte can be quantified with acceptable accuracy and precision.
Based on Signal-to-Noise Approach:-
This approach can only be applied to analytical procedures that exhibit baseline noise. Determination of the signal-to-noise ratio is performed by comparing measured signals from samples with known low concentrations of analyte with those of blank samples and by establishing the minimum concentration at which the analyte can be reliably quantified. A typical signal-to-noise ratio is 10:1. Based on the Standard Deviation of the Response and the Slope The quantitation limit (QL) may be expressed as:
QL =    10 σ 
S
where,

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

The slope S may be estimated from the calibration curve of the analyte. The estimate of σ may be carried out in a variety of ways for example: Based on Standard Deviation of the Blank Measurement of the magnitude of analytical background response is performed by analyzing an appropriate number of blank samples and calculating the standard deviation of these responses.
Based on the Calibration Curve:-
A specific calibration curve should be studied using samples, containing an analyte in the range of QL. The residual standard deviation of a regression line or the standard deviation of y-intercepts of regression lines may be used as the standard deviation.

Recommended Data:-
The quantitation limit and the method used for determining the quantitation limit should be presented. The limit should be subsequently validated by the analysis of a suitable number of samples known to be near or prepared at the quantitation limit.

Linearity & Range:-

A linear relationship should be evaluated across the range of the analytical procedure.  It may be demonstrated directly on the drug substance ( by dilution of a standard stock solution ) and /or separate weighings of synthetic mixtures of the drug product components, using the proposed procedure. The latter aspect can be studied during the investigation of the range.