Routine method, validation

The "method of standard additions" has been employed as a technique for standardization of atomic absorption analyses of metals In biological fluids (13,21) In this procedure, several concentrations of standard analyte are added to samples of the biological fluid to be analyzed The calibration curve which Is obtained after additions of the standard analyte to the biological fluid should parallel that obtained when aqueous standards are analyzed Extrapolation of the standard additions curve back to a negative Intercept on the abscissa furnishes an estimate of the concentration of the analyte In the original sample (21) This technique Is helpful In assessing the validity of methods of trace metal analysis (11,13,58) However, In the author s opinion, the "method of standard additions" Is neither practical nor reliable as a routine method for standardization... 

Other considerations could include availability of reagent(s) or equipment, method for routine analyses vs limited samples, and confirmatory method vs multi-residues. Plan for method validation and/or analytical quality control. 

Better guidance for applicants seems necessary, with the objective that information about extraction efficiency is routinely considered in method validation studies. 

The analyte stability during storage and processing of samples or in standard solutions and extracts is not part of method validation in Germany. Therefore, insufficient stability will not be routinely detected and even then more or less only by chance. Also, separate tests for analyte homogeneity and extraction efficiency were not performed. 

Validate routine methods, i.e., define the conditions under which the assay results are meaningful.115 To do that, one must select samples that are truly representative of the product stream. This may be a difficult task when the process is still under development and the product stream variable. The linearity of detector response should be defined over a range much broader than that expected to be encountered. Interference from the sample matrix and bias from analyte loss in preparation or separation often can be inferred from studies of linearity. Explicit detection or quantitation limits should be established. The precision (run-to-run repeatability) and accuracy (comparison with known standards) can be estimated with standards. Sample stability should be explored and storage conditions defined. 

The approaches described above give approximate values for the LoD and LoQ. This is sufficient if the analyte levels in test samples are well above the LoD and LoQ. If the detection limits are critical, they should be evaluated by using a more rigorous approach [1, 2, 14]. In addition, the LoD and LoQ sometimes vary with the type of sample and minor variations in measurement conditions. When these parameters are of importance, it is necessary to assess the expected level of change during method validation and build a protocol for checking the parameters, at appropriate intervals, when the method is in routine use. 

Method validation provides information concerning the method s performance capabilities and limitations, when applied under routine circumstances and when it is within statistical control, and can be used to set the QC limits. The warning and action limits are commonly set at twice and three times the within-laboratory reproducibility, respectively. When the method is used on a regular basis, periodic measurement of QC samples and the plotting of these data on QC charts is required to ensure that the method is still within statistical control. The frequency of QC checks should not normally be set at less than 5% of the sample throughput. When the method is new, it may be set much higher. Quality control charts are discussed in Chapter 6. 

Note (4) is very important as it highlights the fact that the reference material used for the method validation cannot be used again when the method is in routine use for calibration purposes. The same type of material can be used, but it needs to come from a different supplier. The same material cannot be used for calibration purposes and then as a quality control material. 

Many of the more established techniques have been validated through collaborative studies which becomes of greater importance as laboratories seek to become accredited via ISO, EN or related systems where the use of official or well validated methods is mandatory. New instrumental techniques are constantly being reported in the literature but it often requires many years before procedures are introduced, validated and then applied within the food industry. Recent techniques that can be included in this category are capillary electrophoresis and liquid chromatography-mass spectrometry (LC-MS). In time procedures based on these techniques will also become accepted as routine methods and are likely to be adopted by some of the official international bodies like the AOAC International, CEN, ISO, etc. 

Application New methods, required for full vahdation and standardization, first prerequisite for IQC and QA Routinely used (validation and/ or standardization methods), recommended within IQC and QA system... 

If there is any doubt about whether 100% of the analyte is presented to the measuring system or that the response of the calibrated system leads to no bias, then the assumptions must be tested during method validation and appropriate actions taken. If a series of measurements of a CRM (not used for calibration) leads to the conclusion that there is significant bias in the observed measurement result, the result should be corrected, and the measurement uncertainty should include the uncertainty of the measurement of the bias. If the bias is considered insignificant, no correction need be made, but measuring the bias and concluding that it is zero adds uncertainty (perhaps the bias was not really zero but is less than the uncertainty of its measurement). One approach to measurement uncertainty is therefore to include CRMs in the batch to be used to correct for bias, and then the uncertainty of estimation of bias, which includes the uncertainty of the quantity value of the CRM, is combined with the within-laboratory reproducibility. In some fields of analysis it is held that routine measurement and correction for bias... 

A discussion about calibration must also include consideration of singlepoint calibration and direct comparison of responses to samples of known and unknown quantities. In each case the linearity of the calibration (i.e., the correctness of taking a ratio of instrument responses) is accepted in routine work. In method validation this assumption must be verified by making a series of measurements in a concentration range near to the range used, and the linear model must be demonstrated to be correct. 

Method validation is the process of confirming that the analytical procedure employed for a specific analysis is suitable for its intended use. Analytical methods need to be validated or revalidated before their introduction into routine use, or whenever the conditions for which the methods have been validated change. 

The overall process of method validation is illustrated in Figure I. However, the extent and scope of validation is governed by the applicability of the method. An in-house procedure requires a less exacting process than a method intended for multi-matrix and/or multi-laboratory use. For the latter methods, a full collaborative trial is necessary and is covered in Chapter 9. However, for many purposes validation is limited to either demonstrating that method performance criteria established during development are met under routine laboratory conditions and/or showing method equivalence (Figure 18). 

Routine methods of analysis are defined as methods of analysis to be used by member states to implement national plans for the control of residues in food-producing animals and their products in compliance with Council Directive 96/23 (2). Routine methods must have been validated by operational laboratories and must fulfill the relevant criteria set out in the annex of Commission Decision 93/256 (5). They may be used for screening and/or confirmatory purposes. 

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 in Table 1. 

Large number of results by methods familiar to users Method validation and uncertainty poorly defined Present approaches do not provide traceable data Ideal when well-established methods are widely used Applicable to all common analytical applications Fewer results, often by specialised methods Methods well characterised with uncertainty budgets Traceability of data well defined and accepted Ideal when problems exist with routine methods Not applicable to method-specific applications... 

The absorption of pantoprazole at 295 nm was used for the quantitative determination, and the method validated and used for the analysis of pantoprazole in its tablets. The results of validation study indicated that the method is linear over the range of 1.0 to 3.0 mg/mL (r= 0.9999). The percent recovery and relative standard deviation were 99.3-101.5 (n=9), and less than 1.0%, respectively. This method can be used for quality control and routine analysis [5],... 

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