Ruggedness final method

During method development (Chapter 9) and validation (Chapter 10), QCs are used for several purposes including checks on precision and accuracy, lower limit of quantitation (LLOQ), recovery and method robustness and ruggedness (Section 9.8.4), as well as stability studies of various kinds (Sections 10.2.7 and 10.2.8), studies of inter-day validation within a specified laboratory and cross-validations in inter-laboratory method transfer (Section 10.2.11). QC samples are also used during method development to assess the final method prior to validation experimental runs that use QCs for this purpose are often referred to as assay prequalifications or pre-study assay evaluations (PSAE). 

Sample preparation techniques vary depending on the analyte and the matrix. An advantage of immunoassays is that less sample preparation is often needed prior to analysis. Because the ELISA is conducted in an aqueous system, aqueous samples such as groundwater may be analyzed directly in the immunoassay or following dilution in a buffer solution. For soil, plant material or complex water samples (e.g., sewage effluent), the analyte must be extracted from the matrix. The extraction method must meet performance criteria such as recovery, reproducibility and ruggedness, and ultimately the analyte must be in a solution that is aqueous or in a water-miscible solvent. For chemical analytes such as pesticides, a simple extraction with methanol may be suitable. At the other extreme, multiple extractions, column cleanup and finally solvent exchange may be necessary to extract the analyte into a solution that is free of matrix interference. 

The final evaluation of the analytical procedure with generated filters resulted in a modified procedure which was "rugged" for the analysis of Be, Cd, Co, Cr, Cu, Mn, Mo, Ni, and Pb. The method was not ruggedized for Pd because the absence of perchloric acid from the developed procedure made it incompatible for Pd. 

Ruggedness/robustness Defined based on an experimental design and data (sensitive parameters and a range for each parameter in the final test method)... 

Finally a validation step for the overall MISPE procedure is mandatory to allow the use of the method itself in place of the regulatory methods. The issues to be considered here are the accuracy and precision of the method based on the MISPE protocol, its limit of quantification, selectivity and ruggedness. In particular the inter- and intra-assay precision need to be checked with real samples and certified reference materials and methods [23,24]. 

The results of the ruggedness testing and bias evaluation should be published in full. This report should identify the critical parameters, including the materials within the scope of the method, and detail the effect of variations in these on the final result. It should also include the values and relevant uncertainties associated with bias estimations, including both statistical and reference material uncertainties. Since it is a requirement of the validation procedure that this information should be available before carrying out the collaborative study, publishing it would add little to the cost of validating the method and would provide valuable information for future users of the method. 

During the prestudy evaluation phase, an attempt should be made to evaluate the variety of conditions that may reflect the execution and performance of the method during the in-study phase. The final conditions should be clearly documented in the analytical procedures prior to in-study sample analysis. As an example, robustness assessment could include incubation time tolerances, while ruggedness assessment could include changes in analysts and batch size (Table 4.7). Most robustness and ruggedness evaluations are empirical in nature however, more formal evaluations can also be used [29]. 

Later, Lawrence and Niedzwiadek [37] modified the method to improve its performance (repeatability, ruggedness, recovery) for routine regulatory purposes. Earlier chromatographic conditions (gradient and flow) were changed to reduce the analysis time. The oxidation reaction conditions were modified to achieve the best compromise possible for all the PSP toxins, since it has been shown that the optimum pH for the oxidation of individual toxins varies considerably [38]. A matrix modifier (blank oyster extract) was added to improve the yield and repeatability for the periodate derivatization of standards and sample extracts. Finally, the performance of the second cleanup with SPE-COOH cartridges, which enables the separation of the sulfocarbamoyl toxins from their carbamoyl counterparts, was reevaluated. 

The method should be assessed using the same or similar calibration standards, QCs and blanks that will be used for validation. In addition to a final assessment of sensitivity, linearity, accuracy and precision, additional experiments designed to assess analyte stability during the sample preparation and analysis processes, method robustness and ruggedness, carryover, and potential... 

Tightening of Procedures. This activity has been in progress since December 1989. The first step was to collect procedures from all the participants. The compiled procedures were studied by the participants. Finally, for each method, a recommended procedure (incorporating technical improvements) was developed by the participants. The same process was carried out in each of the participating countries. Currently, these procedures are subjected to ruggedness testing. 

---