METHOD VALIDATION OF ANALYTICAL PROCEDURES

 

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ABOUT AUTHORS
Prakash Chanda Gupta
QC Executive,
National Healthcare Pvt. Ltd., Nepal
p_c_gupta@yahoo.com

ABSTRACT
After the development of an analytical procedure, it is must important to assure that the procedure will consistently produce the intended a precise result with high degree of accuracy. The method should give a specific result that may not be affected by external matters. This creates a requirement to validate the analytical procedures. The validation procedures consists of some characteristics parameters that makes the method acceptable with addition of statistical tools.

REFERENCE ID: PHARMATUTOR-ART-2304

PharmaTutor (ISSN: 2347 - 7881)

Volume 3, Issue 1

Received On: 06/11/2014; Accepted On: 17/11/2014; Published On: 01/01/2015

How to cite this article: PC Gupta; Method Validation of Analytical Procedures; PharmaTutor; 2015; 3(1); 32-39

INTRODUCTION
Validation of an analytical procedure is the process by which it is established, by laboratory studies, that the performance characteristics of the procedure meet the requirements for the intended analytical applications.[1] Method validation provides an assurance of reliability during normal use, and is sometime referred to as “the process for providing documented evidence that the method does what it is intended to do.” The main objective of the validation is to demonstrate that the analytical method is suitable for its intended purpose, is accurate, specific and precise over the specified range that an analyte will be analyzed. Analytical Method Validation is to be performed for new analysis methods or for current methods when any changes are made to the procedure, composition of the drug product and synthesis of the drugs substances.

Common types of analytical procedure that can be validated [2]

  • Identification tests;
  • Quantitative tests for impurities content;
  • Limit tests for the control of impurities;
  • Quantitative tests of the active moiety in samples of drug substance or drug product or other selected component(s) in the drug product.

Typical validation characteristics which should be considered are listed below: [3]

  • Accuracy
  • Precision
  • Specificity
  • Detection Limit
  • Quantitation Limit
  • Linearity
  • Range
  • Robustness

The validation characteristics are to be evaluated on the basis of the type of analytical procedures.

Table 1: Evaluation of Validation Characteristics

Characteristics

Type of Analytical Procedures

Identification

Impurities

Quantitative Tests

Quantitative

Limit

Accuracy

Not evaluated

Evaluated

Not evaluated

Evaluated

Precision

Not evaluated

Evaluated

Not evaluated

Evaluated

Specificity

Evaluated

Evaluated

Evaluated

Evaluated

Detection Limit

Not evaluated

Not evaluated

Evaluated

Not evaluated

Quantitation Limit

Not evaluated

Evaluated

Not evaluated

Not evaluated

Linearity

Not evaluated

Evaluated

Not evaluated

Evaluated

Range

Not evaluated

Evaluated

Not evaluated

Evaluated

Methods and Terminology

1.   Accuracy
The accuracy of an analytical method is the closeness of the test results obtained by that method to the true value.[3] This is sometimes termed trueness. It is recommended that accuracy should be determined using a minimum of nine determinations over a minimum of the three concentration levels, covering the specified range (3 concentrations/3 replicates each of total analytical procedures).[4]

It is measured as the percent of analyte recovered by assay. The recovery can be determined by the equation:

Recovery = Analytical Result x 100%
                   True Value

The recovery should be in the range of Control limit.

The following method can be applied for calculating the Upper Control Limit (UCL) and Lower Control Limit (LCL). The method involves the moving range, which is defined as the absolute difference between two consecutive measurements (|xi-xi-1|). These moving range are averaged and used in the following formulae: [5]

Where, xi is the individual analytical result, is the sample mean, and d2 is a constant commonly used for this type of chart and is based on the number of observations associated with the moving range calculation. Where n = 2 (two consecutive measurements), as here, d2 = 1.128

2. Precision
The precision of an analytical method is the degree of agreement among individual test results when the method is repeated to multiple samplings of a homogeneous sample.[6] The precision of an analytical procedure is usually expressed as the standard deviation or relative standard deviation (coefficient of variation) of a series of measurements.It is indicated by Relative Standard Deviation, RSD, which is determined by the equation:

Where xi is an individual measurement in a set of n measurement and  is the arithmetic mean of the set. Generally, the RSD should not be more than 2%.

2.1  Repeatability
Repeatability refers to the use of the analytical procedure within a laboratory over a short period of time using the same analyst with the same equipment.[3] Repeatability should be assessed using a minimum of nine determinations covering the specified range for the procedure (i.e., three concentrations and three replicates of each concentration or using a minimum of six determinations at 100% of the test concentration).[4]

2.2  Reproducibility
Reproducibility expresses the precision between laboratories (collaborative studies, usually applied to standardisation of methodology). Reproducibility is usually demonstrated by means of an inter-laboratory trial. [7]

2.3  Intermediate Precision
Intermediate precision is the results from within lab variations due to random events such as different days, different analysts, different equipment, etc.[8]

The standard deviation, relative standard deviation (coefficient of variation) and confidence interval should be reported for each type of precision investigated.

3. Specificity
Specificity is the ability to measure accurately and specifically the analyte of interest in the presence of other components that may be expected to be present in the sample matrix such as impurities, degradation products and matrix components. It must be demonstrated that the analytical method is unaffected by the presence of spiked materials (impurities and/or excipients).

In case of identification tests, the method should be able to discriminate between compounds of closely related structures which are likely to be present. Similarly, in case of assay and impurity tests by chromatographic procedures, specificity can be demonstrated by the resolution of the two components which elute closest to each other.[9]

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.

4.  Linearity
Linearity is the ability of the method to elicit test results that are directly, or by a well-defined mathematical transformation, proportional to analyte concentration within a given range.[10] It should be established initially by visual examination of a plot of signals as a function of analyte concentration of content. If there appears to be a linear relationship, test results should be established by appropriate statistical methods. Data from the regression line provide mathematical estimates of the degree of linearity. The correlation coefficient, y-intercept, and the slope of the regression line should be submitted.

It is recommended to have a minimum of five concentration levels, along with certain minimum specified ranges. For assay, the minimum specified range is from 80% -120% of the target concentration.[11]

Regression line, y = ax + b

Where, a  is the slope of regression line and b is the y- intercept.

Here, x may represent analyte concentration and y may represent the signal responses.

Correlation Coefficient,

Where xi is an individual measurement in a set of n measurement and is the arithmetic mean of the set, yi is an individual measurement in a set of n measurement and  is the arithmetic mean of the set.

5. Detection Limit and Quantitation Limit
The Detection Limit is defined as the lowest concentration of an analyte in a sample that can be detected, not quantified. The Quantitation Limit is the lowest concentration of an analyte in a sample that can be determined with acceptable precision and accuracy under the stated operational conditions of the analytical procedures.[12] Some of the approaches to determine the Detection Limit and Quantitation Limit are: [13]

a. Visual Evaluation
Visual evaluation may be used for non-instrumental methods. For non-instrumental procedures, the detection 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 reliably detected. And 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 determined with acceptable accuracy and precision.Visual Evaluation approach may also be used with instrumental methods.

b. Signal to Noise
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 establishing the minimum concentration at which the analyte can be reliably detected for the determination of Detection Limit and reliably quantified for the determination of Quantitation Limit. A signal-to-noise ratio between 3 or 2:1 is generally considered acceptable for estimating the detection limit and A typical signal-to-noise ratio is 10:1 is considered for establishing the quantitation limit.

c. Standard Deviation of the response and the Slope.

The Detection Limit may be expressed as:

DL = 3.3σ/ s

The Quantitation Limit may be expressed as:

QL = 10σ/ s

Where, σ is standard deviation of the response and s is slope of the linearity curve.

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

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