HPLC Method Development & Method Validation (mr.s)

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METHOD DEVELOPMENT AND METHOD VALIDATION FOR THE ESTIMATION OF DRGS BY USING RP-HPLC

Vikas College Of Pharmacy

BY @@@ Mr.S @@@ Pharmaceutical Analysis & Quality

Assurance, Vikas College Of Pharmacy, Vissannapeta.

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CONTENTS Introduction Principle Method Development * Method Development * Optimization

Techniques Method Validation * Method Validation * Validation ParametersConclusion References

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INTRODUCTI

ON

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INTRODUCTION High Performance Liquid Chromatography

(HPLC) is the most widely used of all analytical separation techniques.

It is a technique in analytical chemistry used to separate the components in a mixture , to identify each component, and to quantify each component.

It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material.

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Each component in the sample interacts slightly differently with the adsorbent material , causing different flow rates for the different components and leading to the separation of the components as they flow out the column.

The development of HPLC from classical column chromatography can be attributed to the development of smaller particle is important.

They offer more surface area over the conventional larger particle sizes.

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PRINCIPLE

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PRINCIPLE “ The principle of separation in normal phase

mode and reversed phase mode is adsorption. When a mixture of components are introduced into a HPLC column, they travel according to their relative affinities towards the stationary phase, more affinity towards stationary phase travels slower and less affinity towards the stationary phase travels faster. No two components have the same affinity towards the stationary phase, the components are separated.”

In most of the cases in pharmaceutical industries –RP-HPLC is used. Hence this gives detailing about RP-HPLC.

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METHOD DEVELOPMENT

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METHOD DEVELOPMENT HPLC method development and

validation play important role in the discovery, development and manufacture of pharmaceutical products, agro chemicals.

HPLC method should be developed within the GMP and GLP environments using the protocols set out in ICH guide lines.

HPLC method development includes: * Method Development * Need for development * Selection of suitable

method * Optimization

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STEPS INVOLVED IN METHOD DEVELOPMENT:- Information on sample Define separation goals Special procedure requirement & pretreatment. Detector selecting and setting Optimization separation conditions Check for problems or needs of special procedure Recovery of purified material Quantitative calibration / Qualitative method Method validation for release to laboratories.

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NEED FOR DEVELOPING A NEW METHOD Several reasons are available for the

development of a new method of analysis, they are :

There may not be a suitable method for a particular analyte in the sample matrix.

Existing may be too erroneous. Existing method may not provide adequate

sensitivity. Existing methods may be unreliable. Existing methods are too expensive and time

consuming.

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SELECTION OF SUITABLE METHOD Using the available literature and

previous methodology, the methods are adopted and modified.

Sample preparation and instrument conditions are adopted to make use of latest methods and instrumentation.

If no previous methods exist for the analyte in the literature , work from analogy to investigate compounds that are similar in structure and properties.

Usually a compound with analytical method exists that is similar to the sample of interest.

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OPTIMIZATION The principle of optimization

in the method development is to reduce the cost, time and also to minimize the errors which are arising in the development.

The process of optimization includes:

* Selection of method * Selection of mobile phase * Selection of column * Selection of detector

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SELECTION OF METHOD The most commonly used

chromatographic methods are normal phase, reverse phase, reverse phase ion pair and ion exchange methods.

In the selection of method for the organic compounds primarily reverse phase method should be tried.

If not successful normal phase should be tried, then reverse phase ion pair method , ion exchange chromatographic methods are at the end.

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SELECTION OF COLUMN Columns being the heart of HPLC

for optimum separation method. The selection of column involves

the following parameters: * Column length * Column diameter * Column particle size * Column particle shape

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SELECTION OF MOBILE PHASE In reversed phase chromatography

the selection of mobile phase is very important for the analysis of the drug .

The organic phase concentration required for the mobile phase can be estimated by “gradient elution method.”

Separation can be optimized by changing the initial mobile phase composition according to the chromatogram obtained from preliminary run.

The pH of the mobile phase should not alters the pH of the sample.

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SELECTION OF DETECTORS Detectors are the eyes of the

chromatography system and measure the compounds after separation of the column.

The must have certain characteristics i.e, high Sensitivity, higher linear range, application to most of the solutes,does not contribute to band broadening, non-destructive, faster response.

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METHOD VALIDATION

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METHOD VALIDATION Validation Establish a documented evidence which

provides a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications and quality attributes.

Method validation is defined as the process of proving (through scientific studies) analytical method is acceptable for its intended use.

The validation of analytical procedures is directed to the four most common types of analytical procedures:

Identification tests, Quantitative tests for impurity content, Limit tests for the control of impurities, Quantitative tests of the active moiety in

samples.

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NEED OF METHOD VALIDATION Before introduction of a new method in to

routine use. Whenever condition change for which

method has been validation e.g. instrument with different characteristics.

Whenever the method is changed and the change is outside the scope of the original method.

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DISCRIPTION OF METHOD VALIDATION

PROCEDURES INVOLVED IN VALIDATION: 1.Accuracy. 2.Precision. 3.Specificity/selectivity. 4.Limit of Detection. 5.Limit of Quantification. 6.Linearity. 7.Robustness. 8.System suitability.

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ACCURACY Accuracy The accuracy is the closeness of the test

results obtained by the method to he true value. Accuracy should be established across its range. Accuracy assessed using a minimum of 9

determinations over a minimum of 3 concentration levels The acceptance criterion for accuracy is the Relative

Standard Deviation (RSD) for all the recovery values should not be more than 2%.

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PRECISION & REPEATABILITY Precision : The precision of an analytical method is

the degree of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample. The RSD for all the assays of sample preparations should not be more than 2%.

Repeatability : Repeatability expresses the precision under the same operating conditions over a short interval of time. Repeatability is also termed intra-assay precision . a minimum of 9 determinations covering the specified range for the procedure ( e.g., 3 concentrations/3 replicates each); or a minimum of 6 determinations at 100% of the test concentration.

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SPECIFICITY Specificity/Selectivity: The ability to assess

unequivocally the sample in the presence of components that may be expected to be present. – Impurities – degrading agents – excipients , Specificity must be demonstrated for: – Identification – Impurities Test – Assay Test.

The RSD for all the assays of sample preparations should not be more than 2%.

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LINEARITY Linearity: The linearity of an analytical procedure is

its ability to obtain test results that are directly proportional to the concentration of the analyte in the sample. Linearity is usually demonstrated by the analysis of various concentrations of the analyte (s) across the indented range and represented graphically.

The relationship between the concentration (in %) of drug in sample area of should be linear in the specified range and the correlation should not be less than 0.9.

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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 use. The RSD for the samples should not be more than 2%.

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System suitability: The simplest form of an HPLC system suitability test involves a comparison of chromatogram trace with a standard trace.

This allows a comparison of the peak shape, peak width, baseline resolution. Parameters to be calculated to provide a system suitability test report.

Parameter LimitCapacity factor K’>2Injection Precision RSD < 1% for n ≥ 5Resolution R>2Tailing factor A≤2Theoritical plates N>2000

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PARAMETERS INVOLVED IN VALIDATION 1.Mean. (Xi) 2.Standard deviation.(S.D) 3.Relative standard deviation.

(RSD) 4.Correlation co-Efficient.(R) 5.Linear regression.

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VALIDATION PARAMETERSMEAN : The average result (ā) is

calculated by summing the individual results and dividing the sum by the number (n) of individual values.

Xi= x1 + x2 + x3 ........ /n Where, x1 ,x2 ,x3...... = values of individual

results n = Number of individual results.

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STANDARD DEVIATION It is the root mean square deviation of values

from their average SD = [Σ (X- Xi) /n-1]1/2 Where, Σ = sum of observations Xi = mean X = individual observation value (X- Xi) = deviation of a value from

mean n =number of observations

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RELATIVE STANDARD DEVIATION

Relative Standard Deviation (RSD) is defined as the standard deviation expressed as the percentage of mean

RSD = (SD/XI) × 100 Where, SD = Standard Deviation XI = Mean

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CORRELATION CO-EFFECIENT The correlation co-efficient (R) is used

to estimate the relationship of two random variables.

It provides a measure of the strength and direction of the correlation varying from +1 to -1.

Positive values indicate the two variables are positively correlated, meaning the two variables vary in the same direction .

Negative values indicate that the two variables are negatively correlated, meaning the two variables vary in the contrary direction.

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Values close to +1 or -1 reveal the two variables are highly related

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LINEAR REGRESSION A regression is a statistical

analysis assessing the association between any two variables. It is used to find the relationship between two variables.

y = a + b x where , a = Intercept b = Slope

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CONCLUSION Whenever the method is changed and the

change is outside the scope of the original method.

If no previous methods exist for the analyte in the literature , work from analogy to investigate compounds that are similar in structure and properties.

HPLC method should be developed within the GMP and GLP environments using the protocols set out in ICH guide lines.

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REFERENCES

Instrumental methods of chemical analysis By B.K. Sharma (Pg:286-385)

Instrumental methods of Chemical Analysis By Gurudeep R Chathwal (Pg: 2.624-2.639)

WIKIPEDIA.

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Science

HPLC Method Development & Method Validation (mr.s)