ANALYTICAL METHODANALYTICAL METHODVALIDATION FOR RSVALIDATION FOR RS

Bhanu Prakash . NAnalytical R&D

Email: bhanu.analytical@gmail.com

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.

Analytical Method Validation

• Different Types of Validation characteristics

Precision Accuracy Specificity Linearity Range Detection Limit Quantitation Limit 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

validation parameter,system suitability criteria can be established.

Acceptance Criteria :

As per the test method.

Analytical Method Validation

• Precision :

• Definition :

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

applied repeadtly 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.

Analytical Method Validation Methodology :

1. Prepare the impurity stock solution of known concentration (individual / Mix). While preparation of stock solution consider the purity of analytes, target concentrations and concentration matrix.

2. Spike the portion of the stock solution to test sample ( Drug product /Drug substance) so that the concentration of the impurity would be 0.3%(or specification limit) of the test concentration as per the test method.(100% level).

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

Acceptance Criteria:

Relative standard deviation; NMT 15.0%

Analytical Method validation:

• Accuracy:

Definition:

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

Methodology for Accuracy :

1. Prepare the impurity stock solution of known concentration (individual/Mix).

2. Spike the known portion of the stock solution to test sample ( Drug product/Drug substance) so that the concentration of the impurity would be 0.3% (or specification limit) of the test concentration as per the test method.(100% level).

Analytical Method validation:

Methodology for Accuracy Cont….. :

3. In the same manner as above, spike the impurity solution at different levels e.g 50%,75%,125%,150% (or specified in protocol) so that the concentration of the impurity will be 0.15%,0.225%,0.375% and 0.45% respectively, of the test concentration.Perform the analysis as per the test method and calculate the % recovery.

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

Acceptance Criteria: % Recovery should be NLT 85.0% and NMT 115.0%.

Analytical Method Validation

• Detection Limit:

Definition:

It is lowest amount of analyte in a sample that can be detected but not

necessarily quantitated under the stated experimental conditions.

Methodology:

Following are different approaches

1) Visual Evaluation Method.

Prepare the sample solutions with known lowest possible concentrations

of analyte and establish the minimum concentration at which the analyte

can be reliably detected by analysing as per test method .

Analytical Method Validation

2) Based on Signal to Noise Ratio Method.

Prepare the analyte solutions with known lowest possible concentrations

of analyte and analyse as per test method .

Consider the concentration as a Limit of detection which gives signal to noise ratio

between 3 : 1 or 2 : 1

3) Based on the standard Deviation of the Response and the Slope:

Prepare the blank solution as per test method and inject six times into the chromatographic system.

Similarly prepare the linearity solution staring from lowest possible concentration of analyte to 150% (or as per protocol) of target concentration and establish the linearity curve.

3) Based on standard deviation and of the.

Analytical Method Validation

The detection limit (DL) may be expressed as

3.3 x standard deviation of the response of the blank

DL = ----------------------------------------------------------------------------

Slope

The slope shall be estimated from the calibration curve of the

analyte.

Analytical Method Validation

• Quantitation Limit:

Definition:

It is lowest amount of analyte in a sample which can be quantitatively

determined with acceptable accuracy and precision.

Methodology:

Following are different approaches

1) Visual Evaluation Method.

Prepare the sample solutions with known lowest possible concentrations

of analyte and establish the minimum concentration at which the analyte

can be reliably quantified by analysing as per test method .

Analytical Method Validation2) Based on signal to noise ratio method.

Prepare the analyte solutions with known lowest possible concentrations of

analyte and analyse as per test method .

Consider the concentration as a Limit of Quantification which gives signal

to noise ratio between 10 : 1.

3) Based on the standard Deviation of the Response and the Slope:

Prepare the blank solution as per test method and inject six times into the chromatographic system.

Similarly prepare the linearity solution staring from lowest possible concentration of analyte to 150% (or as per protocol) of target concentration and establish the linearity curve.

Analytical Method Validation

The Quantification limit (QL) may be expressed as

10 x standard deviation of the response of the blank

QL = ----------------------------------------------------------------------------

Slope

The slope shall be estimated from the calibration curve of the

analyte.

Perform the Precision and accuracy at the level of limit of quantification by

spiking LOQ concentration on placebo / Drug

product / Drug substance.

For detail methodology and acceptance criteria refer Precision and accuracy of

test method.

Analytical Method Validation

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. 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 20%.

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. Prepare the sample and placebo solution as per test method and analyse.

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.

Acceptance Criteria :

1. Placebo / Blank should not elute at the retention time of analyte peak.2. Peak purity of analyte peak should be confirmed.3. Degradation of active analyte peak should be from 1% to 20%.

Analytical Method Validation

• 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 limit of quantification level to 150% (or as per protocol) of the target concentration and establish the calibration curve by analysing the sample as per test method.

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

Analytical Method Validation

• 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 0.997.

Analytical Method Validation

• 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.

Analytical Method Validation

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.

Analytical Method ValidationMethodology :

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.

Acceptance Criteria :

For Variations : 1. System suitability should meet the acceptance criteria as per test method. 2. RRT’s of impurity peaks should be comparable with RRT’s mentioned in test method.

For filter suitability : Difference between the % impurity should not be more than 0.04 against the unfiltered / centrifuged sample.

Analytical Method Validation

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.

Analytical Method ValidationMethodology :

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.

Acceptance Criteria :

The % Difference should not be more than 0.04% for individual known impurity and 0.1% for total impurities from initial value.

Similarity factor of standard should be NLT 0.95 and NMT 1.05.

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