The document discusses analytical method development and validation for the simultaneous estimation of active pharmaceutical ingredients (APIs). It covers topics such as the need for method development, the steps involved, parameters for validation as outlined in ICH guidelines, and the use of high performance liquid chromatography (HPLC). The key objectives of validation are to demonstrate that the developed method is suitable for its intended purpose and can reliably quantify the APIs.

1. ANALYTICAL METHOD DEVELOPMENT AND
VALIDATION FOR SIMULTANEOUS ESTIMATION of api
Prepared By:
S. L. Khan
Asst. Professor
Pharmaceutical Chemistry
Ali-Allana College of Pharmacy,
Akkalkuwa

2. CONTENTS
1. INTRODUCTION
2. ANALYTICAL METHOD DEVELOPMENT
3. METHOD VALIDATION
4. OBJECTIVES OF VALIDATION
5. HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
6. CONCLUSION
7. REFERENCES
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3. 1.INTRODUCTION
 Analytical chemistry deals with the separation, quantification and chemical components
identification of natural and artificial materials constituted with one or more compounds
or elements
 Analytical chemistry is separated into two main categories:
1) Qualitative Analysis 2) Quantitative Analysis
 Pharmaceutical analysis plays outstanding role in the examination of pharmaceutical
formulations and bulk drugs regarding the quality control and assurance
 Rapid increase in pharmaceutical industries and production of drug in and around the
world cause increase in demand to seek novel and systematic analytical techniques in the
pharmaceutical industries
 As a result, analytical method development has become the basic activity of analysis
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4. Contd…
 Development in scientific and concrete analytical methods has been resulted from the
advancements of analytical instruments
 The improvements of the analytical method development and analytical instruments
have reduced the time and cost of analysis and enhanced precision and accuracy
 Techniques applicable to analysis are developed and validated for active pharmaceutical
ingredients, excipients, related substances, drug products, degradation products and,
residual solvents, etc
 Resulting which become an integral part of the required necessities for regulatory
organization
 Analytical method development results in official test methods
 Quality control laboratories used these methods to check the efficacy, identity, purity,
safety and performance of the drug products
 Drug approval by regulatory authorities requires the applicant to prove control of the
entire process of drug development by using validated analytical methods
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5. 2.ANALYTICAL METHOD DEVELOPMENT
 When no authoritative methods are available, new methods are being developed for
analysis of novel products
 To analyze the existing either pharmacopoeial or non-pharmacopoeial products, novel
methods are developed to reduce the cost besides time for better precision and ruggedness
 These methods are optimized and validated through trial runs
 Purpose of analytical method development…
1) When there is no official drug or drug combination available in the pharmacopoeias
2) When there is no decorous analytical process for the existing drug in the literature due to
patent regulations
3) When there are no analytical methods for the formulation of the drug due to the
interference caused by the formulation excipients
4) Analytical methods for the quantitation of the analyte in biological fluids are found to be
unavailable
5) The existing analytical procedures may need costly reagents and solvents.
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6. Contd…
 Steps for the development of the method
 Analyte standard characterization
 Method requirements
 Literature search and prior methodology
 Choosing a method
 Instrumental setup and initial studies
 Optimization
 Documentation
 Evaluation of development method with real samples
 Estimation of percent recovery of real samples and demonstration of quantitative
sample analysis
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7. VALIDATION:
“Validation is a documented evidence of the consistency of the any process OR system. It
was firstly appeared in 1978 in the US by FDA to investigate the failure of the product in
sterility and now it is very common concept in pharmaceutical industries through out the
world.”
 The process of validation of analytical method is adopted to confirm that the employed
analytical procedure for a specific tests meet the intended requirements
 Guidelines from the USP, ICH, FDA etc., can provide a framework for validations of
pharmaceutical methods
 Results from the method validation can be considered to judge its quality, reliability as
well consistency applicable to analytical results
3.METHOD VALIDATION
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8.  Parameters of Analytical Method Validation
Analytical methods have been validated in pursuance of ICH guidelines of Q2 (R1).
Validation parameters are:
1) System suitability
2) Specificity
3) Linearity
4) Precision
5) Accuracy
6) LOD [limit of detection]
7) LOQ[limit of quantification]
8) Robustness
9) Ruggedness
Contd…
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9. 4.Objectives of Validation
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10. Chromatography is the most widely used laboratory technique for separation identification
and quantification of components of liquid and gaseous mixtures. Solid mixtures are also
analysed by first converting them to a liquid or gaseous state, using suitable sample
preparation techniques.
Principle
High performance liquid chromatography (HPLC) is separation technique utilizing
differences in distribution of compounds to two phases; called stationary phase and mobile
phase. The stationary phase designates a thin layer created on the surface of fine
particles a mobile phase designates the liquid flowing over particles. Under a certain
dynamic condition, each component in a sample has difference distribution equilibrium
depending on solubility in the phases and or molecular size. As a result, the components
move at different speed over the stationary phase and thereby separated from each other.
5. High Performance Liquid Chromatography
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11. Calibration
“The demonstration that a particular instrument or device produces results within
specified limits by comparison with those produced by a reference or traceable standard
over an appropriate range of measurements”.
Calibration of HPLC:
Various Calibration parameters are:
 Flow rate accuracy
 Injector accuracy
 Wavelength accuracy
 Injector linearity
 System Precision
 Detector linearity
 Column oven temperature accuracy
Contd…
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12. Types of detectors
A detector gives specific response for the components separated by the column and also
provides the required sensitivity. It has to be independent of any changes in mobile phase
composition. Majority of the applications require UV-VIS detection though detectors
based on other detection technique are also popular these days
• UV-VIS detector
• Photo diode array detector
• Fluorescence detector y detector
• Mass spectroscopic detector
• Refractive index detector
• Electrochemical detector
• Light scattering detectors
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13. Schedule for Calibration/Inspection of Some Major
Instruments:
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6. CONCLUSION
 When analytical method is utilized to generate results about the characteristics of
drug related samples it is essential that the results are trustworthy
 They may be utilized as the basis for decisions relating to administering the drug to
patients
 Analytical method validation required during drug development and manufacturing
and these analytical methods are fit for their intended purpose
 To comply with the requirements of GMP pharmaceutical industries should have an
overall validation policy which documents how validation will be performed
 The purpose of this validation is to show that processes involved in the development
and manufacture of drug, production and analytical testing can be performed in an
effective and reproducible manner

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6. REFERENCES
1. G. David Watson, Pharmaceutical Analysis (3rd Ed., Churchill Livingstone, London:
Harcourt Publishers Limited, Essex CM 20 2JE, 2012).
2. A.H. Beckett, and J.B. Stenlake, Practical Pharmaceutical Chemistry (4th Ed., Vol. I &
II. CBS Publishers and Distributors, New Delhi: 2007).
3. T. Higuchi, and Brochman-Hansen, Pharmaceutical Analysis, (3rd edition, CBS
Publishers and Distributors pvt. Ltd., New Delhi:1997).
4. Lloyd R.Snyder,Joseph J.Kirkland, Joseph L.Glanjch.Practical, HPLC method
development, A Wiley – interscience publication,1997.
5. Y.Anjaneyulu, R.Marayya, Quality Assurance and Quality Management in
Pharmaceutical industry, Pharmabook Syndicate, 2005.
6. ICH Guidelines Q2BValidation of Analytical Procedures: Methodology (1996).

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