Procedures for Method Validation

The validity of a specific method should be demonstrated in laboratory experiments using test samples or standards that are similar to the unknown samples analyzed in the routine. For example, control tissue samples from non-treated animals, control tissue samples spiked with the analyte at several known concentrations, and dosed or incurred tissue samples from animals that have been treated with the drug, should all be available to the analyst before starting a validation process. All these samples will enable the analyst to define the background noise, to identify the amounts of the analyte added to the control tissue, and demonstrate that the method can satisfactorily recover the biologically incurred residue. 

The preparation and execution should follow a validation protocol, in which the scope of the method and its validation criteria should first be defined (46). The scope of the analytical method should be clearly understood since this will govern the validation characteristics that need to be evaluated. For example, if the method is to be used for qualitative trace residue analysis, there is no need to examine and validate its linearity over the full dynamic range of the equipment. The scope of the method should also include the different types of equipment and the locations where the method will be run. In this way, experiments can be limited to what is really necessary. For example, if the method is intended for use in one specific laboratory, there is no need to include other laboratories and different equipment in the validation experiments. 

On the other hand, a complete list of validation criteria should be agreed on by the developer and the end users before the method is developed, so that expectations are becoming clear. During the acmal smdies and in the final valida- 

Satisfactory results for a method can only be obtained with well-performing equipment. Therefore, before an instrument is used to validate a method, its performance should be verified using universal standards (47). Special attention should be paid to the equipment specifications that are critical for the performance of the method. For example, if detection limit is critical for a specific method, the detector specifications for baseline noise and the response to the specified compounds should be checked. Furthermore, any reagent or reference standard used to determine critical validation parameters should be double-checked for accurate composition and purity. 

The suitability of a method for its intended use should be proven in initial experiments. These introductory studies should include the approximate precision, accuracy, detection limit, and working range. If these preliminary validation data appear to be inappropriate, either the method itself or the acceptance limits should be changed. The developer does not know whether the method conditions are acceptable until validation studies are performed. Results of validation studies may indicate that a change in the procedure is necessary, which may then require revalidation. In this way, method development and validation seems to be an iterative process during each developmental phase, key method parameters are determined and then used for all subsequent validation steps. 

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METHOD CHARACTERISTICS AND TEST PROCEDURES FOR METHOD VALIDATION... 

Validated analytical methodology. Analytical methods are implemented only if they have been correctly validated. The general procedure for method validation is defined in the several ISO (International Standards Organisation) 17025 companion documents. The accumulation of a vast body of analytical data is most often necessary covering both intra- and interlaboratory variations. WADA is the ultimate body that allows the use of a newly... 

No universal method for validation exists, but several standard operation procedures for method validation can be found in the literature and from regulatory agencies such as ISO/IEC 17025 and FDA CGMP. 

One method that is often nsed for method validation is the addition of a surrogate to the sample matrix, then following the track of the surrogate through the separation procedure. It is appropriate at this point, to comment on such a procedure. 

ICH Guidelines for Method Validation Q2B Validation of Analytical Procedures Methodology, 1996, http //www.ich.org/pdfICH/Q2B.pdf. 

FDA Draft Guidance for Industry on Analytical Procedures and Methods Validation Chemistry, Mamrfacturing, and Controls Docirmentation, 2000, http //www.fda.gov/... 

Guidance for Industry, Analytical Procedures and Method Validation Chemistry, Manufacturing and Controls Documentation, Draft, August 2000, Center for Drug Evaluation and Research, Center for Biologies Evaluation and Research, FDA, Department of Health and Human Services, 2000. 

Guidance for Industry, Analytical Procedures and Methods Validation, Chemistry Manufacturing and Controls Documentation, Draft Guidance, FDA, August 2000. 

U.S. Food and Drug Administration (FDA) (2000), Guidance for industry, Analytical procedures and methods validation—Chemistry, manufacturing, and controls documentation, FDA, Rockville, MD. 

Easy and standard procedure for method development, validation, and sample analysis ... 

The tests and methods used by the applicant for acceptance of each batch of a packaging component that they receive should be described. If a batch is to be accepted based on a supplier s COA or COC, then the procedure for supplier validation should be described. The data from the supplier s COA or COC should clearly indicate that the lot meets the applicant s acceptance criteria. Acceptance criteria for extractables should also be included, if appropriate. 

As already mentioned, the analytical parameters required for method validation and for the estimation of measurement uncertainty can be evaluated without assigned values. But to assess the accuracy of delivered results, as stated in ISO/IEC 17025, there is a requirement for assigned values with a stated uncertainty, which are traceable to the same reference as the analytical results of the method used. In physics, measurements are made in accordance with the SI units, which were introduced under the convention of the meter. In chemical measurements, traceability of results to SI units is not always possible. Therefore, the role of chemical standards is decisive in establishing the comparability of results between laboratories. During the validation of the analytical procedure, traceability of the result can be demonstrated by comparison against the certified value of a CRM, which provides exactly this traceable assigned value with a stated uncertainty. 

Once an analytical method (e.g., LC/MS/MS) is established, it is necessary to qualify or validate the procedure from a regulatory GLP perspective. The desired criteria for method validation/qualification include determining the lower and upper LOQ, inter- and intraday precision, specificity of the method, and linearity of the calibration curves (166). Validation/qualification must be performed in the presence of the representative biological matrix that will be used in reaction phenotyping. For CYP reaction phenotyping studies, the matrix of choice is a pool of human liver microsomes (166). 

The work in one central laboratory could be considered for the study of sampling techniques, in particular for environmental or workplace hygiene problems. Finally, a new project using the same approach allowed to set up a standardised procedure for the validation of microbiological methods, compared to a reference method. 

Guidance for Industry Analytical Procedures and Methods Validation Chemistry, Manufacturing, and Controls Documentation. Food and Drug Administration Rockville MD, 2000. 

Figure 8.1.5 Procedure for development and implementation of NORMAN protocols for method validation...

Before implementation of analytical methods for routine use, careful validation is required to demonstrate that the method is suitable for the intended purpose. Analytical methods employed for the quantitative determination of drug substances and their metabolites in biological media play a significant role in the evaluation and interpretation of pharmacokinetic data. To define the requirements and procedures for the validation of bioanalytical methods, a conference was held in 1990 in Crystal City in the Washington, DC, area, which was co-sponsored by the U.S. Food and Drug Administration (FDA), The Canadian Health Protection Branch, the American Association of Pharmaceutical Scientists (AAPS), and the Association of Official Analytical Chemists (AOAC), bringing together scientists from... 

Analytical methods must also be validated. The approach used for method validation is consistent with the validation of analytical methods for drug products. See USP monograph on analytical validation procedures. 

USP <1225> [3] is another useful source for method validation information. This chapter is specific to compendial procedures but its use for all types of methods... 

Methods validation would be critically reviewed for completeness, accuracy and reliability. If an alternative to a compendial method is used, this in-house method must be compared to the compendial method to demonstrate it is equivalent or superior to the official procedure. Appropriate pharmacopoeial guidelines for methods validation should be followed. 

Validation of pharmaceutical CE methods requires similar considerations to those examined during HPLC validation procedures. A useful guide for method validation in analytical chemistry is the... 

ICH Q2(R1) Recommendations for Method Validation of Analytical Test Procedures... 

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