Introduction to Validation of Analytical Methods
The process of analytical method validation should demonstrate that the method is fit for its purpose. The validation should follow a plan that includes the scope of the method, the method performance characteristics and acceptance limits.
Parameters usually examined in the validation process are limits of detection and quantitation, accuracy, precision, selectivity/specificity, linearity, range and ruggedness. A validation report should be generated with all experimental conditions and the complete statistics. If standard methods are used, it should be verified that the scope of the method and validation data, for example, sample matrix, linearity, range and detection limits comply with the laboratory’s analyses requirements, otherwise the validation of the standard method should be repeated using the laboratory’s own criteria. The present article gives a brief review on analytical method validation.
Method validation is the process by which it is established that performance characteristics of the method meet the requirements for the intended analytical applications. Methods need to be validated or revalidated before their introduction into routine use.1 The International Conference on Harmonization (ICH) of Technical Requirements for the Registration of Pharmaceuticals for Human Use2 has developed a text on the validation of analytical procedures. The United States Food and Drug Administration (USFDA) have proposed guidelines on submitting samples and analytical data for methods validation5-7. The United States Pharmacopoeia (USP) has published specific guidelines for method validation for compound evaluation8. The document includes definitions for eight validation characteristics. An extension with more detailed
methodology is in preparation and nearly completed3. The United States Environmental Protection Agency (US EPA) prepared a guidance for methods development and validation for the Resource Conservation and Recovery Act (RCRA)4. The pharmaceutical industry uses methodology published in the literature 9,10. The most comprehensive document was published as the ‘Conference Report of the Washington Conference on Analytical Methods Validation: Bioavailability, Bioequivalence and Pharmacokinetic Studies held in 1990 (sponsored by the American Association of Pharmaceutical Scientists, the AOAC and the US FDA, among others)10. The report presents guiding principles for validation of studies in both human and animal subjects that may be referred to in developing future formal guidelines. Representatives of the pharmaceutical and chemical industry have published papers on the validation of analytical methods. Hokanson 11,12 applied the life cycle approach, developed for computerized systems, to the validation and revalidation of methods. Green13 gave a practical guide for analytical method validation with a description of a set of minimum requirements for a method. Renger and his colleagues 14 described the validation of a specific analytical procedure for the analysis of theophylline in a tablet using high performance thin layer chromatography (HPTLC). The validation procedure in that article is based on requirements for European Union multistate registration. Wegscheider 15 has published procedures for method validation with special focus on calibration, recovery experiments, method comparison and investigation of ruggedness. The Association of Official Analytical Chemists (AOAC) 16 has developed a Peer-Verified Methods validation program with detailed guidelines on what parameters should be validated. This article gives a review and a strategy for the validation of analytical methods for both in-house developed as well as standard methods and a recommendation on the documentation that should be produced during and at the end of method validation.
Strategy for Validation of Methods
The validity of a specific method should be demonstrated in laboratory experiments using samples or standards that are similar to the unknown samples analyzed in the routine. The preparation and execution should follow a validation protocol, preferably written in a step by step instruction format. Possible steps for a complete method validation are listed below.
Steps in Method Validation
1. Develop a validation protocol or operating procedure for the Validation
2. Define the application, purpose and scope of the method
3. Define the performance parameters and acceptance criteria
4. Define validation experiments
5. Verify relevant performance characteristics of equipment
6. Qualify materials, e.g. standards and reagents
7. Perform pre-validation experiments
8. Adjust method parameters or/and acceptance criteria if necessary
9. Perform full internal (and external) validation experiments
10. Develop SOPs for executing the method in the routine
11. Define criteria for revalidation
12. Define type and frequency of system suitability tests and/or analytical quality control (AQC) checks for the routine
13. Document validation experiments and results in the validation.
First the scope of the method and its validation criteria should be defined. These include: Compounds, matrices, type of information, qualitative or quantitative, detection and quantitation limits, linear range, precision and accuracy, type of equipment and location. The scope of the method should include the different types of equipment and the locations where the method will be run. The method’s performance characteristics should be based on the intended use of the method. For example, if the method will be used for qualitative trace level analysis, there is no need to test and validate the method’s linearity over the full dynamic range of the equipment. Initial parameters should be chosen according to the analyst’s best judgment. Finally, parameters should be agreed between the lab generating the data and the client using the data. Instruments performance should be verified using generic standards, before an instrument is used to validate a method.18, 19. These studies should include the approximate precision, working range and detection limits. If the preliminary validation data appear to be inappropriate, either the method itself or the equipment or the analysis technique or the acceptance limits should be changed. In this way method development and validation is an iterative process. For example, in liquid chromatography selectivity is achieved through selection of mobile phase composition. For quantitative measurements the resolution factor between two peaks should be 2.5 or higher. If this value is not achieved, the mobile phase composition needs further optimization. There are no official guidelines on the sequence of validation experiments and the optimal sequence can depend on the method itself.
A validation report should be prepared that includes:
• Objective and scope of the method (applicability, type)
• Type of compounds and matrix
• Detailed chemicals, reagents, reference standards and control sample preparations
• Procedures for quality checks of standards and chemicals used
• Safety considerations
• Method parameters
• Critical parameters indicated from robustness testing
• Listing of equipment and its functional and performance requirements, e.g. cell
dimensions, baseline noise, column temperature range
• Detailed conditions on how the experiments were conducted, including sample
• Statistical procedures and representative calculations
• Procedures for quality control in the routine (e.g., system suitability tests)
• Representative plots, e.g. chromatograms, spectra and calibration curves
• Method acceptance limit performance data
• The expected uncertainty of measurement results
• Criteria for revalidation
• Person who developed and initially validated the method
• Summary and conclusions
Validation of Standard Methods
A laboratory applying a specific method should ensure that they have documentary evidence that the method has been appropriately validated. “The responsibility is with the user to ensure that the validation documented in the method is sufficiently complete to meet his or her needs.”1 When standard methods are used, their scope should be in line with the scope of the laboratories, method requirements and the suitability of the entire analytical system in the specific laboratory‘s environment should be verified for the method. The laboratory should demonstrate the validity of the method in the laboratories environment. Full validation of a standard method is recommended where no information on type and results of validation can be found in the standard method documentation.
A revalidation is necessary whenever a method is changed and the new parameter is outside the operating range. Operating ranges should be defined for each method based on experience with similar methods, or they should be investigated during method developments. These ranges should be verified during method validation in robustness studies and should be part of the method characteristics. Availability of such operating ranges makes it easier to decide when a method should be revalidated. If, for example, the operating range of the column temperature has been specified to be between 30 and 40°C, if, for whatever reason, the new operating parameter has been selected as 41°C, then the method should be revalidated. Revalidation is also required if the sample matrix changes and if the instrument type changes.
Parameters For Method Validation:
The parameters as defined by the ICH2, 3 and by other organizations and authors are Specificity, selectivity, precision, repeatability, intermediate precision, reproducibility, accuracy, trueness, bias, linearity range, limit of detection, limit of quantitation, robustness and ruggedness.
Selectivity / Specificity
The terms selectivityand specificity are often used interchangeably. A detailed discussion of this term as defined by different organizations has been made by Vessmann 17. Even inconsistent with ICH, the term specific generally refers to a method that produces a response for a single analyte only, while the term selective refers to a method which provides responses for a number of chemical entities that may or may not be distinguished from each other. If the response is distinguished from all other responses, the method is said to be selective. Since there are very few methods that respond to only one analyte, the term selectivity is usually more appropriate. The USP monograph 8 defines selectivity of an analytical method as its ability to measure accurately an analyte in the presence of interference, such as synthetic precursors, excipients, enantiomers and known (or likely) degradation products that may be expected to be present in the sample matrix.
Determination:-In the case of qualitative analyses, the ability to select between compounds of closely related structure that are likely to be present should be demonstrated. This should be confirmed by obtaining positive results from samples containing the analyte, coupled with negative results from samples that do not contain the analyte and by confirming that a positive response is not obtained from materials structurally similar to or closely related to the analyte.23
Selectivity in liquid chromatography is obtained by choosing optimal columns and setting chromatographic conditions such as mobile phase composition, column temperature and detector wavelength. It is a difficult task in chromatography to ascertain whether the peaks within a sample chromatogram are pure or consist of more than one compound. While in the past chromatographic parameters such as mobile phase composition or the column has been modified. More recently the applications of spectroscopic detectors coupled on-line to the chromatograph have been suggested3, 5 The principles of diode-array detection in HPLC and their application and limitations to peak purity are described in the literature 20-22.
Precisions and Reproducibility
The precision of a method is the extent to which the individual test results of multiple injections of a series of standards agree. The measured standard deviation can be subdivided into three categories: repeatability, intermediate precision and reproducibility2, 3.
Repeatability is obtained when one operator using one piece of equipment over a relatively short time-span carries out the analysis in one laboratory. At least 5 or 6 determinations of three different matrices at two or three different concentrations should be done and the relative standard deviation calculated.
Intermediate precision is a term that has been defined by ICH2 as the long-term variability of the measurement process and is determined by comparing the results of a method run within a single laboratory over a number of weeks.A method’s intermediate precision may reflect discrepancies in results obtained by different operators, from different instruments, with standards and reagents from different suppliers, with columns from different batches or a combination of these.
Objective of intermediate precision validation is to verify that in the same laboratory the method will provide the same results once the development phase is over.
Reproducibility as defined by ICH2, 3 represents the precision obtained between laboratories. Objective is to verify that the method will provide the same results in different laboratories.
Typical variations affecting a method’s reproducibility
• Differences in room temperature and humidity
• Operators with different experience and thoroughness
• Equipment with different characteristics, e.g. delay volume of an HPLC system
• Variations in material and instrument conditions, E.g. in HPLC, mobile phases
composition, pH, flow rate of mobile phase
• Equipment and consumables of different ages
• Columns from different suppliers or different batches
• Solvents, reagents and other material with different quality
Accuracy and recovery
The accuracy of an analytical method is the extent to which test results generated by the method and the true value agree. The true value for accuracy assessment can be obtained in several ways.
Determination:-In case of a drug substance, accuracy may be determined by application of the analytical method to an analyte of known purity or by comparison of the results with well characterized method ,the accuracy of which has been stated or defined.23
Linearity and calibration curve
The linearity of an analytical method is its ability to elicit test results that are (directly or by means of well-defined mathematical transformations) proportional to the concentration of analytes in samples within a given range. Linearity is determined by a series of three to six injections of five or more standards whose concentrations span 80-120 percent of the expected concentration range. The response should be (directly or by means of a well-defined mathematical calculation) proportional to the concentrations of the analytes. A linear regression equation applied to the results should have an intercept not significantly different from zero. If a significant nonzero intercept is obtained, it should be demonstrated that there is no effect on the accuracy of the method.
The range of an analytical method is the interval between the upper and lower levels (including these levels) that have been demonstrated to be determined with precision, accuracy and linearity using the method as written. The range is normally expressed in the same units as the test results (e.g. percentage, parts per million) obtained by the analytical method.
The range of the 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.
Limit of Detection and Quantitation
Limit of detection: It is the lowest concentration of analyte in a sample that can be detected but not necessarily quantified. In chromatography the detection limit is the injected amount that results in a peak with a height at least twice or three times as high as the baseline noise level.
Determination:-the detection limit is generally determined by the analysis of samples with known concentration of analyte and by establishing the minimum level at which the analyte can be reliably detected.23
Limit of quantitation:It is the minimum injected amount that gives precise measurements , in chromatography typically requiring peak heights 10 to 20 times higher than baseline noise.
Ruggedness is measure of reproducibility test results under the variation in conditions normally expected from laboratory to laboratory and from analyst to analyst.
The Ruggedness of an analytical method is degree of reproducibility of test results obtained by the analysis of the same samples under a variety of conditions, such as; different laboratories, analysts, instruments, reagents, temperature, time etc.23
Robustness of analytical method 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.
1. EURACHEM Guidance Document No. 1/WELAC Guidance Document No. WGD 2: Accreditation for chemical laboratories: Guidance on the interpretation of the EN 45000 series of standards and ISO/IEC Guide 25,1993. Available from the EURACHEM Secretariat, PO Box 46, Teddington, Middlesex, TW11 ONH, UK.
2. International Conference on Harmonization (ICH) of Technical Requirements for the Registration of Pharmaceuticals for Human Use, Validation of analytical procedures, ICH-Q2A, Geneva 1995.
3. International Conference on Harmonization (ICH) of Technical Requirements for the Registration of Pharmaceuticals for Human Use, Validation of analytical procedures: Methodology, ICH-Q2B, Geneva 1996.
4. US EPA, Guidance for methods development and methods validation for the Resource Conservation and Recovery Act (RCRA) Program, Washington,1995.
5. US FDA Technical Review Guide: Validation of Chromatographic Methods, Center for Drug Evaluation and Research (CDER), Rockville, MD, 1993.
6. US FDA, General principles of validation, Rockville, MD, Center for Drug Evaluation and Research (CDER), May 1987.
7. US FDA, Guidelines for submitting samples and analytical data for method validation, Rockville, MD, Center for Drugs and Biologics Department of Health and Human Services , Feb. 1987.
8. General Chapter <1225>, Validation of compendial methods,United States Pharmacopeia XXIII, National Formulary, XVIII, Rockville, MD, The United States Pharmacopeial Convention, Inc, 1995, 1710–1612.
9. Szepesi, M. Gazdag and K. Mihalyfi, Selection of HPLC methods in pharmaceutical analysis -III method validation, www.labcompliance.com Page 21 J.Chromatogr. 464, 265-278.
10. P. Shah et al., Analytical methods validation: Bioavailability, bioequivalence and pharmacokinetic studies, Eur. J. Drug Metabolism and Pharmacokinetics, 16(4),249-255, 1989 (1991).
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12. G.C. Hokanson, A life cycle approach to the validation of analytical methods during pharmaceutical product development, part II: Changes and the need for additional validation, Pharm.Tech., Oct. 1994, 92-100.
13. J.M.Green, A practical guide to analytical method validation, Anal.Chem. News & Features, May 1, 1996, 305A/309A 14. B. Renger, H.Jehle, M.Fischer and w. Funk, Validation of analytical procedures in pharmaceutical analytical chemistry: HPTLC assay of theophylline in an effervescent tablet, J.Planar Chrom., 8, July/Aug 1995, 269-278.
15. Wegscheider, Validation of analytical methods, in “Accreditation and quality assurance in analytical chemistry”, edited by H. Guenzler, Springer Verlag, Berlin 1996.
16. AOAC Peer Verified methods Program, Manual on policies and procedures, Arlington, VA, Nov 1993.
17. J.Vessman, Selectivity or specificity ? Validation of analytical methods from the perspective of an analytical chemist in the pharmaceutical industry, J.Pharm&Biomed Analysis, 14 (1996) 867/869.
18. Huber, L. “Validation of computerized analytical systems, Part 3: Installation and operational qualification”; LC-GC Magazine 1996, 14(9), 806-812 www.labcompliance.com Page 22.
19. L. Huber, Validation of Computerized Analytical Systems, Interpharm, Buffalo Grove,IL, 1995.
20. Huber, Applications of diode-array detection in HPLC, Waldbronn, Germany, Agilent Technologies, 1989, publ. number 12-5953-2330.
21. D. Marr, P. Horvath, B. J. Clark, A. F. Fell, Assessment of peakhomogeneity in HPLC by computer-aided photodiode-array detection, Anal. Proceed.,23, 254-257, 1986.
22. Huber and S. George, Diode-array detection in highperformanceliquid chromatography, New York, Marcel Dekker, ISBN 0-8247-4, 1993.
23. General Chapter <1225>, Validation of compendial methods, United States Pharmacopeia, Twenty-Sixth Revision, National Formulary, Twenty-first Edition, Rockville, MD, The United States Pharmacopeial Convention, Inc, 2003,2440-2442.
Mr.Sohan S.Chitlange (M.Pharm. Pharmaceutical Chemistry)
Asst. Professor, Dept.of Pharm.Chemistry, Padm. Dr.D.Y. Patil Institute of Pharmaceutical Sciences & Research, Pimpri, Pune.
Mr. Amol A. Kulkarni(M.Pharm. Pharmaceutical Chemistry)
Lecturer, Dept.of Pharm.Chemistry, Padm. Dr.D.Y. Patil Institute of Pharmaceutical Sciences & Research, Pimpri, Pune.
Mohammed Imran (M.Pharma. Sem.II Quality Assurance)