Method Validation

To avoid errors due to inexperience, the validation experiments should be carried out by an experienced analyst. And also the analyst should be very well versed in the technique and operation of the instrument. Instrument performance specifications are verified using generic chemical standards before an instrument is used to validate a method. Satisfactory results can be obtained for a method only with equipment that is performing well. Special attention should be paid to equipment characteristics that are critical for the method. For example, if detection limit is critical for a specific method, the instrument’s specification for baseline noise and, for certain detectors, the response to specified compounds should be verified.

Any chemical such as reagents and reference standards used to determine critical validation parameters should be available in sufficient quantities, accurately identified, sufficiently stable and checked for exact composition and purity. Any other materials and consumables, for example, chromatographic columns, should be new and be qualified to meet the column’s performance criteria. This ensures that one set of consumables can be used for most experiments. When operators are sufficiently familiar with the technique and equipment it will allow them to identify and diagnose unforeseen problems more easily and to run the entire process more efficiently.

If there is little or no information on the method’s performance characteristics, it is recommended to prove the suitability of the method for its intended use in initial experiments. These studies should include the approximate precision, working range and detection limits. If the preliminary validation data appear to be inappropriate, the method itself, the equipment, the analysis technique or the acceptance limits should be changed. Method development and validation are, therefore, an iterative process. For example, in liquid chromatography, selectivity is achieved through the 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. The influence of operating parameters on the performance of the method will be assessed at this stage if this was not done during development and optimization of the method.

There are no official guidelines on the correct sequence of validation experiments, and the optimal sequence will depend on the method itself. For a liquid chromatographic method, the following sequence has proven to be useful while validating: Selectivity of standards (optimizing separation and detection of standard mixtures if selectivity is insufficient) Linearity, limit of quantitation, limit of detection, range Repeatability (short-term precision) of retention times and peak areas Intermediate precision Selectivity with real samples Trueness/accuracy at different concentrations Ruggedness (inter laboratory studies) The more time-consuming experiments, such as accuracy and ruggedness, are generally included toward the end. Some of the parameters, as listed under (2) to (6), can be measured in combined experiments.

For example, when the precision of peak areas is measured over the full concentration range, the data can be used to validate the linearity also. During method validation, the parameters, acceptance limits and frequency of ongoing system suitability tests or quality control checks should be defined. At the same time criteria should be defined to indicate when the method and system are beyond statistical control. Hence the aim is to optimize these experiments, with a minimum number of control analyses, the method and the complete analytical system will provide long-term results to meet the objectives defined in the scope of the method. Once the method has been developed and validated, a validation report should be prepared including objective and scope of the method (applicability, type), summary of methodology, type of compounds and matrix, all chemicals, reagents, reference standards, QC samples with purity, grade, their source or detailed instructions on their preparation, procedures for quality checks of standards and chemicals used, safety precautions, a plan and procedure for method implementation from the method development lab to routine analysis, method parameters, critical parameters taken from robustness testing, listing of equipment and its functional and performance requirements, e.g., cell dimensions, baseline noise and column temperature range.

For complex equipment, a picture or schematic diagram may be useful, detailed conditions on how the experiments were conducted, including sample preparation. The report must be detailed enough to ensure that it can be reproduced by a competent technician with comparable equipment, statistical procedures and representative calculations, procedures for QC in routine analyses, 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, the person(s) who developed and validated the method, references, summary and conclusions, approval with names, titles, date and signature of those responsible for the review and approval of the analytical test procedure.