Assay-Method validation-PPT _slide

Bhanu Prakash. N(Analytical R&D)

email: [email protected]

Analytical Method Validation for

Assay

Analytical Method Validation

What is Validation ?

The Objective of validation of an analytical method is to demonstrate

that it is suitable for it’s intended purpose.

OR

It is the process by which it is established, by laboratory studies, that

the performance characteristics of the method meet the requirements

for the intended analytical applications.


• Different Types of Validation characteristics

Precision Accuracy Specificity Linearity Range Ruggedness Robustness

Analytical Method validation

• Considerations & requirements prior to Method Validation

Instruments / Equipments are validated and calibrated.

Reference Standards and Reagents are standardized.

Analysts are trained and qualified.

Availability of Test methods, glassware's etc.

Availability of test material.

Approved protocols.

Analytical Method validation System Suitability :

System suitability testing is an integral part of 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.

System suitability test parameters to be established for a particular

procedure depend on the type of procedure being validated.

Analytical Method validation• Methodology :

Prepare the solution and evaluate system suitability parameters as per the test method .

Based on the evaluation of system suitability parameters at

each validation parameter,system suitability criteria can be

established.

Acceptance Criteria :

As per the test method.


• Precision :

• Definition :

It is degree of agreement among individual test results when the method is

applied repeatedly to multiple samplings of a homogeneous sample.

• The precision of test method is usually expressed as the standard deviation or

relative standard deviation of a series of measurements.

• It is a measure of either the degree of reproducibility or repeatability of the

analytical method under normal operating conditions.


Methodology :

1. Prepare the sample as per the test method and analyze.

2. Perform the the analysis on six determinations at 100% level of test

concentration.

Acceptance Criteria:

Relative standard deviation; NMT 2.0%

Analytical Method validation:

• Accuracy:

Definition:

It is the closeness of agreement between true value and measured value.

Methodology for Accuracy :

1. If the test method is dropping of tablet ,spike the active ingredient from 50%,75%,100%,125% and 150% of the test concentration with placebo equivalent to the test concentration(Subtract the weight of active ingredient) and perform the analysis.

2. In case of crushing tablet method ,weigh the sample equivalent to 50%,75%,100%,125% and 150% of the test concentration and perform the

analysis.

Analytical Method validation:

Methodology for Accuracy Cont….. :

3 .Perform the analysis on three different determinations at each spike level..

Acceptance Criteria: % Recovery should be NLT 97.0% and NMT 103.0%.


Specificity :

Definition :

The ability to assess unequivocally the analyte in the presence of components

that may be expected to present, such as impurities, degradation products and

matrix components.

Methodology:

Specificity shall be demonstrated by performing Placebo / blank interference

and forced degradation studies.

Analytical Method Validation Methodology cont…..:

1. Blank interference :

Prepare blank solution as per test method and analyse as per test method.

2. Placebo interference (In case of Drug products) :

Prepare the placebo solution equivalent to the test concentration (Subtract the weight of active ingredient) and analyse as per test method.

3. Spike the known impurities to the test preparation at 0.3% or specification limit level.

4. Force Degradation studies : Degrade the sample forcefully under the various stress conditions like Light, heat, humidity, acid/base/water hydrolysis and oxidation and ensure the degradation from 1% to 50%.

Analytical Method ValidationMethodology Cont……:

a) Light : Expose the Drug product, drug substance and placebo to UV light for about 200watts/M2 and 1200 Klux for visible light.

b) Heat : Expose the Drug product, drug substance and placebo at 105 °C for about 12 hours. Prepare the sample and placebo solution as per test method and analyse.

c) Humidity : Expose the Drug product, drug substance and placebo for about 80% RH at about 25°C for about one week. Prepare the sample and placebo solution as per test method and analyse.

d) Acid /Base : Prepare the acid or base solution of 0.1N and reflux the sample and placebo with 50ml of acid/base solution for about 1 hour at 60°C. Neutralize the solution and dissolve the contents in diluent as per test method. Change the strength of acid and base or refluxion time to ensure the desired degradation.

Analytical Method ValidationMethodology Cont….:

e) Water : Reflux the sample / placebo with 100ml water for 12 hour at 60°C Dissolve the contents in diluent as per test method. Change the refluxion time so as to ensure the desired degradation

f) Oxidation : Reflux for 12 hour at 60°C. Dissolve the contents in diluent as per test method. Change the refluxion time so as to ensure the desired degradation.

Note : Based on the physio-chemical properties and literature stress conditions can be decided.


1. Peak purity of analyte peak should be confirmed.2. Degradation of active analyte peak should be from 1% to 50%.


• Linearity :

Definition:

The linearity of an analytical method is it’s ability to elicit test results that are directly or by a well defined mathematical transformation, proportional to the concentration of the analyte in a sample within the given range.

Methodology :

Prepare the analyte solutions from 50% to 150% (or as per protocol) of the target concentration and establish the calibration curve by analyzing the sample as per test method.

Linearity shall be established across the range. Minimum five concentration across the range are recommended.


• Linearity cont…… Calculate the co-efficient of co-relation, Y-intercept and slope of regression

line.

If linearity is not meeting the acceptance criteria, establish the range of concentration in which it is linear.

Acceptance criteria:

Co efficient of co relation should be NLT 0.999.


• Range :

The range of an analytical method is the interval between the upper and lower levels of analyte that have been demonstrated to be determines with a suitable level of precision, accuracy and linearity using the test method.

The range of the test method is validated by verifying that the analytical method provides acceptable precision, accuracy and linearity when applied to samples containing analyte at the extremes of the range as well as within the range.

Range can be established during linearity and accuracy study,

Acceptance Criteria:

Same as applicable for Precision, Linearity and accuracy.


Robustness :

Definition: It is a measure of method's ability to remain unaffected by small but

deliberate variations in method parameters.

The following are the typical method parameters need to change deliberately and verify during method validation :

Flow rate (+/- 0.2ml/minutes) Mobile phase composition (+/- 10% of organic phase) Column oven temperature (+/- 5°C) pH of buffer in mobile phase (+/- 0.2 units) Filter suitability (At least two filters)

Analytical Method ValidationMethodology :

a) Mobile phase variation: Prepare the mobile phases by changing organic phase to ±10% of the mobile phase composition.

b) Flow rate : Change the flow rate by ± 0.2 ml/minutes of the target flow rate mentioned in test method.

c) Temperature of the Column : Change the temperature the column by ± 5.0°C of the target temperature mentioned in Test method.

d) pH of the buffer of mobile phase : Prepare the mobile phases by changing the pH of buffer by ± 0.2 units of the pH mentioned in the test method.


E) Filter Suitability : Prepare the test solution as per test method and filter through two different types filters. Analyse the sample as per test method and compare the results against unfiltered / centrifuged sample.


For Variations : 1. System suitability should meet the acceptance criteria as per test method.

For filter suitability : Similarity factor should be from 0.98-1.02


Ruggedness :

Definition: It is a measure of method reproducibility under variable conditions within

specified test parameters of test method.

The following are the typical method parameters need to tested during method validation :

Analyst-to-Analyst variability. Column-to-Column variability. System-to-System variability. Different days. Different Laboratories. Stability of Solutions and mobile phase. (At least for 48 hours)

Analytical Method Validation• Methodology :

• Perform the precision as mentioned in precision of test method by using different systems, different columns, different analyst , in different Laboratories and on different day.

• The method is to be consider rugged for individual variation, if it meets the precision of test method and method transfer acceptance criteria.


Perform the bench top and refrigerator stability of the standard and test solutionfor 24hrs and 48 hrs ,calculate the results each time against freshly preparedstandard solution.If 24hrs stability is not meeting the acceptance criteria,perform the stability studies on hourly basis on bench top and in refrigerator.


The % Difference should not be more than 2.0% for an assay from initial value.

Similarity factor of standard should be NLT 0.98 and NMT 1.02.

THANK YOU

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Assay-Method validation-PPT _slide