To avoid errors due toinexperience, the validation experiments should be carried out by anexperienced analyst. And also the analyst should be very well versed in thetechnique and operation of the instrument. Instrument performancespecifications are verified using generic chemical standards before aninstrument is used to validate a method. Satisfactory results can be obtained fora method only with equipment that is performing well. Special attention shouldbe paid to equipment characteristics that are critical for the method. Forexample, if detection limit is critical for a specific method, the instrument’sspecification for baseline noise and, for certain detectors, the response tospecified compounds should be verified. Anychemical such as reagents and reference standards used to determine criticalvalidation parameters should be available in sufficient quantities, accurately identified, sufficiently stable and checked for exact composition and purity.
Any other materials andconsumables, for example, chromatographic columns, should be new and bequalified to meet the column’s performance criteria. This ensures that one setof consumables can be used for most experiments. When operators are sufficientlyfamiliar with the technique and equipment it will allow them to identify anddiagnose unforeseen problems more easily and to run the entire process moreefficiently. Ifthere is little or no information on the method’s performance characteristics,it is recommended to prove the suitability of the method for its intended usein initial experiments.
These studies should include the approximate precision,working range and detection limits. If the preliminary validation data appearto be inappropriate, the method itself, the equipment, the analysis techniqueor the acceptance limits should be changed. Method development and validationare, therefore, an iterative process. For example, in liquid chromatography,selectivity is achieved through the selection of mobile phase composition.
Forquantitative measurements, the resolution factor between two peaks should be2.5 or higher. If this value is not achieved, the mobile phase compositionneeds further optimization. The influence of operating parameters on the performanceof the method will be assessed at this stage if this was not done duringdevelopment and optimization of the method. Thereare no official guidelines on the correct sequence of validation experiments,and the optimal sequence will depend on the method itself. For a liquid chromatographicmethod, 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-consumingexperiments, such as accuracy and ruggedness, are generally included toward theend. Some of the parameters, as listed under (2) to (6), can be measured incombined experiments.
For example, when the precision of peak areas is measuredover the full concentration range, the data can be used to validate thelinearity also.During methodvalidation, the parameters, acceptance limits and frequency of ongoing systemsuitability tests or quality control checks should be defined. At the same timecriteria should be defined to indicate when the method and system are beyondstatistical control. Hence the aim is to optimize these experiments, with aminimum number of control analyses, the method and the complete analyticalsystem will provide long-term results to meet the objectives defined in thescope of the method.Once the method has beendeveloped and validated, a validation report should be prepared including objectiveand scope of the method (applicability, type), summary ofmethodology, type of compounds and matrix, all chemicals, reagents,reference standards, QC samples with purity, grade, their source or detailed instructionson their preparation, procedures for quality checks of standards and chemicalsused, safety precautions, a plan and procedure for method implementation fromthe method development lab to routine analysis, method parameters, criticalparameters taken from robustness testing, listing of equipment and itsfunctional and performance requirements, e.g.
, cell dimensions, baseline noiseand column temperature range. For complex equipment, a picture or schematicdiagram may be useful, detailed conditions on how the experiments wereconducted, including sample preparation. The report must be detailed enough toensure that it can be reproduced by a competent technician with comparableequipment, statistical procedures and representative calculations, proceduresfor QC in routine analyses, e.g., system suitability tests, representativeplots, e.g., chromatograms, spectra and calibration curves, method acceptancelimit performance data, the expected uncertainty of measurement results,criteria for revalidation, the person(s) who developed and validated themethod, references, summary and conclusions, approval with names, titles, dateand signature of those responsible for the review and approval of theanalytical test procedure.