Analytical Method Validation

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it gives description about parameters to be validated when analytical method is developed.

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Ms. Rashami V. Chaudhari M.Pharm Sem II Quality Assurance R. C. Patel Institute of Pharmaceutical Education and Research

What is Validation?:

What is Validation? Validation is defined as the process of demonstrating that the analytical procedure is suitable for its intended purpose Identification Determination of impurities Assay 15 April 2012 2 Analytical Method Validation

Why Validation?:

Why Validation? The objective of any analytical measurement is to obtain consistent, reliable and accurate data. Validated analytical methods play a major role in achieving this goal. Validation of analytical methods is also required by most regulations. l. 15 April 2012 3 Analytical Method Validation

Typical Analytical Procedures To Be Validated:

Typical Analytical Procedures To Be Validated Four most common types of analytical procedures to be validated: Identification tests Quantitative tests for impurities content Limit tests for the control of impurities Quantitative tests of the active moiety 15 April 2012 4 Analytical Method Validation

Parameters:

Parameters 15 April 2012 5 Analytical Method Validation

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Characteristic Identification Impurities Testing Assay Quantitative Limit Accuracy _ + _ + Precision a. Repeatability _ + _ + b. Intermediate precision _ + _ + Specificity + + + + LOD _ _ + _ LOQ _ + _ _ Linearity _ + _ + Range _ + _ + signifies that this characteristic is not normally evaluated + signifies that this characteristic is normally evaluated 15 April 2012 6 Analytical Method Validation

Revalidation:

Revalidation Revalidation may be necessary in the following circumstances: Changes in the synthesis of the drug substance Changes in the composition of the finished product Changes in the analytical procedure 15 April 2012 7 Analytical Method Validation

1. Linearity and Range:

1. Linearity and Range Ability to obtain test results that are directly (or by a well-defined mathematical transformation) proportional to the concentration of analyte in samples within a given range. The range of an analytical procedure is the interval between the upper and lower levels of analyte (including these levels) that have been demonstrated to be determined with a suitable level of precision, accuracy, and linearity 15 April 2012 8 Analytical Method Validation

Determination of Linearity and Range:

Determination of Linearity and Range For establishment of linearity, minimum 5 concentrations are recommended. Linearity results should be established by appropriate statistical methods. 15 April 2012 9 Analytical Method Validation

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Transformations are also acceptable and may include log, square root, or reciprocal (other transformations are acceptable) Conc. (µg/ml) Response 1 0.0625 2 0.25 3 0.562 4 0.922 5 1.562 15 April 2012 10 Analytical Method Validation

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If linearity is not attainable, a nonlinear model may be used. The goal is to have a model (whether linear or nonlinear) that describes closely the concentration-response relationship. Conc. (µg/ml) √ Response 1 0.25 2 0.5 3 0.75 4 0.96 5 1.25 15 April 2012 11 Analytical Method Validation

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The following parameters should be determined: correlation coefficient y-intercept slope of the regression line The range of the procedure is validated by verifying that the analytical procedure provides acceptable precision, accuracy, and linearity when applied to samples containing analyte at the extremes of the range as well as within the range. 15 April 2012 12 Analytical Method Validation

2. Precision:

2. Precision The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. 15 April 2012 13 Analytical Method Validation

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Concentration Absorbance 10 µg/ml Mean 0.21 0.32 0.39 0.53 0.36 Less Variation More Variation High Precision Low Precision Concentration Absorbance 10 µg/ml Mean 0.28 0.31 0.29 0.30 0.29 15 April 2012 14 Analytical Method Validation

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Precision may be considered at three levels: 15 April 2012 15 Analytical Method Validation

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Repeatability Repeatability expresses the precision under the same operating conditions over a short interval of time. Repeatability should be assessed using a minimum of 9 determinations covering the specified range. 2. Intermediate Precision Intermediate precision expresses variations within laboratories, such as different days, different analysts, different equipment, and so forth 3. Reproducibility Reproducibility expresses the precision between laboratories. It is assessed by means of an inter-laboratory trial. (Defined as ruggedness in USP, ISO 17025) 15 April 2012 16 Analytical Method Validation

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Following parameters should be reported: Standard deviation Relative standard deviation (coefficient of variation) 15 April 2012 17 Concentration µg/ml Absorbance SD & % RSD 8 0.337 0.00041, 1.223% 0.348 0.341 12 0.575 0.0106, 1.815% 0.583 0.596 20 0.967 0.0091, 0.933% 0.985 0.978 Analytical Method Validation

3. Accuracy:

3. Accuracy Closeness of agreement between the conventional true value / an accepted reference value and the value found. High Accuracy Less Accuracy (Less Precision) (High Precision) 15 April 2012 18 Analytical Method Validation

Determination of Accuracy:

Determination of Accuracy a) application of analytical procedure to a reference material b) to compare the results c) accuracy may be inferred once precision, linearity and specificity have been established. a)application of the analytical procedure to synthetic mixtures to which known quantities of the drug substance have been added b) to compare the results c) accuracy may be inferred once precision, linearity and specificity have been established. Drug Substance Drug Product 1. Assay 15 April 2012 19 Analytical Method Validation

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2. Impurities (Quantitation) Assessed on samples (drug substance/drug product) spiked with known amounts of impurities. Accuracy should be assessed using a minimum of 9 determinations over a minimum of 3 concentration levels covering the specified range (e.g., 3 concentrations/3 replicates each of the total analytical procedure). Accuracy should be 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. 15 April 2012 20 Analytical Method Validation

4. Limit of Detection & Limit of Quantitation:

4. Limit of Detection & Limit of Quantitation Limit of Detection: It is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated. Limit of Quantitation: It is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. 15 April 2012 21 Analytical Method Validation

Determination of LOD & LOQ:

Determination of LOD & LOQ Method Based on visual evaluation Based on standard deviation of response and slope LOD = 3.3 σ / Slope Signal to noise ratio 2:1 or 3:1 Method Based on visual evaluation Based on standard deviation of response and slope LOD = 10 σ / Slope Signal to noise ratio 10:1 Limit of Detection Limit of Quantitation 15 April 2012 22 Analytical Method Validation

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15 April 2012 23 Analytical Method Validation

6. Specificity:

6. Specificity The ability to detect the analyte of interest in the presence of interfering substances (typically impurities, degradants, matrix). 1. 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 from samples containing the analyte, coupled with negative results from samples which do not contain the analyte. 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. 15 April 2012 24 Analytical Method Validation

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2. Assay and impurity test: a. Impurities are available For the assay , this should involve demonstration of the discrimination of the analyte in the presence of impurities and/or excipients. This can be done by spiking pure substances with appropriate levels of impurities and/or excipients and demonstrating that the assay result is unaffected by the presence of these materials. 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. 15 April 2012 25 Analytical Method Validation

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b. 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. As appropriate, this should include samples stored under relevant stress conditions: light, heat, humidity, acid/base hydrolysis and oxidation. 15 April 2012 26 Analytical Method Validation

7. Robustness:

7. Robustness The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. If measurements are susceptible to variations in analytical conditions, the analytical conditions should be suitably controlled or a precautionary statement should be included in the procedure, such as: Use solution within 24 hours Maintain temperature below 25 degrees 15 April 2012 27 Analytical Method Validation

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In the case of liquid chromatography, examples of typical variations are: influence of variations of pH in a mobile phase influence of variations in mobile phase composition different columns (different lots and/or suppliers) temperature flow rate In the case of gas-chromatography, examples of typical variations are: different columns (different lots and/or suppliers) temperature flow rate 15 April 2012 28 Analytical Method Validation

8. System Suitability:

8. System Suitability System suitability testing is an integral part of many analytical procedures. The tests are based on the concept that the equipment, electronics, analytical operations and samples to be analyzed constitute an integral system that can be evaluated as such. 15 April 2012 29 Analytical Method Validation

Conclusion:

Conclusion When the method is properly validated consistent, reliable and accurate results are obtained. Also, Validation of analytical methods is also required by regulations. Hence it is very important to validate any analytical method that has been developed. 15 April 2012 30 Analytical Method Validation

References:

References ICH harmonised tripartite guideline validation of analytical procedures ICH Q2A: Definitions and terminology ICH harmonised tripartite guideline validation of analytical procedures ICH Q2B: Methodology Method Validation in Pharmaceutical Analysis, A guide to best practice, Edited by Joachim Ermer , John H. McB Miller, Wiley- VCH Chung Chow Chan et.al. “Analytical Method Validation and Instrument Performance Verification, Wiley Interscience , A John Wiley & Sons inc. Publication A Primer, Validation of Analytical Methods, Agilent Technologies, Ludwig Huber. United States Pharmacopoeia, The National Formulary-2005, Government of United States http://www.labcompliance.com http://www.agilent.com/chem/pharmaqaqc http://www.fda.gov 15 April 2012 31 Analytical Method Validation

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15 April 2012 32 Analytical Method Validation

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