Reproducibility analytical method

Ourane, R., Guibert, A., Brown, D., and Bomet, F., A sensitive and reproducible analytical method to measure fructo-oligosaccharides in food products, in Complex Carbohydrates Definition, Analysis and Applications, Cho, L., Prosky, L., and Dreher, M., Eds., Marcel Dekker, New York, 1997, pp. 191-201. 

The application of a wide variety of chromatographic techniques to the analysis of additives in biopolymers is a current tendency in many research laboratories around the world. The increasing interest in the use of biopolymers in many technological applications will raise the research in this field in the future. Therefore, the potential of chromatography for separation, identification, and quantification will be very important for the development of reliable and reproducible analytical methods. 

The column is arguably the most important component in HPLC separations. The availability of a stable, high-performance column is essential for developing a rugged, reproducible analytical method. Performance of columns from different vendors can vary widely. Separation selectivity, resolution, and efficiency depend on the type and quality of the column. Proper column maintenance is the key to ensure optimum column performance as well as an extended column lifetime. It ensures stability of column plate number, band symmetry, retention, and resolution. The major issues related to column performance and maintenance are discussed here. 

Several HPLC methods for the assessment of flavins in urine are available, the majority being published before 1989 (for review, see 1) (18,26,33,70,71). HPLC has been shown to be a fast, sensitive, simple, and reproducible analytical method for urinary flavins (Table 5). Using fluorescence detection, flavin concentrations as low as 0.05 to 0.30 Ag/mL and as high as 2 to 12 p,g/mL RF can be measured without concentration or dilution, respectively. No special pretreatment procedure is necessary for HPLC of urine samples. Fresh urine samples are injected either directly or after centrifugation, to remove most of the sediment. Due to bacterial degradation, the stability of riboflavin in fresh urine is limited at room temperature, even in the dark. Consequently, all flavin-containing urine samples should be stabilized by acidification (acetic or oxalic acid) and/or adding a small amount of toluene. 

The EPA publishes Series Methods that describe the exact procedures to be followed with respect to sample receipt and handling, analytical methods, data reporting, and document control. These guidelines must be followed closely to ensure accuracy, reproducibility, and reliability within and among the contract laboratories. 

Two-Dimensional Electrophoresis. Two-dimensional (2D) electrophoresis is unique, offering an analytical method that is both reproducible and sensitive. It is referred to as 2D because it employs two different methods of electrophoresis, in two different dimensions, to produce one result. Each method separates the sample compounds based on different properties of each compound. The combination of the two methods gives better resolution of the compounds in the sample than could be achieved with either method alone. For example, each method alone may separate up to 100 components of a sample, whereas together they may separate up to 10,000 components. 

As noted earlier, the x -test for goodness-of-fit gives a more balanced view of the concept of fit than does the pure least-squares model however, there is no direct comparison between x and the reproducibility of an analytical method. 

Precision The repeatability characterizes the degree of short-term control exerted over the analytical method. Reproducibility is similar, but includes all the factors that influence the degree of control under routine and long-term conditions. A well-designed standard operating procedure permits one to repeat the sampling, sample work-up, and measurement process and repeatedly obtain very similar results. As discussed in Sections 1.1.3 and 1.1.4, the... 

The typical product of a traditional analytical method is a static, paper report. Such reports usually consist of a vast number of discrete, personal, ad hoc processes that cannot typically be used to perform subsequent comprehensive reproducible analyses as soon as additional analyses are needed. Having an... 

Traditional analytical methods make extensive use of computers, but typically these methods still require constant restructuring of the data and multiple analytical tools. This endless restructuring wastes time and productivity and also makes the analytical processes difficult to document, audit, and reproduce in real time. This situation also makes it difficult to reconstruct and update analyses in real time when new adverse event data become available or when new questions need to be asked. The application of comprehensive data standards allows the use of integrated, reusable software for analyzing adverse event data. This integration facilitates the reproducibility of the results. 

The issue of data reproducibility had been the previous focus of the AOAC Official Methods Program. The TDRM will assist AOAC in responding to an expanded scope of it mission to include statements of accuracy with AOAC methods. This extra step requires independent verification of the accuracy of measurements generated by the method. Accuracy of an analytical method must be verified at three separate points ... 

Currently, nutrient analytical methods development often utilizes the method of standard additions as an intrinsic aspect of the development process. Essentially, the analyte to be measured exists in the matrix to which an identical known pure standard is added. The spiked and non-spiked matrix is extracted and analysed for the nutrient of interest. By spiking at increasing levels the researcher can establish, to some degree of certainty, the recovery and linearity of the standard additions. One can also evaluate data to determine reproducibility, precision, and accuracy. Unfortunately, the method of standard additions does not allow the evaluation of the method at nutrient concentrations less than 100 % of the endogenous level. 

Annex VI to Directive 91/414/EEC concerning the placing of plant protection products on the market. The section concerning residue analytical methods was not fully finalized when the Directive was first adopted. There were no provisions for methods to determine residues from a.i. and relevant metabolites in soil, water, and air. The criteria for foodstuffs partly proved to be not helpful for the practice of assessment (e.g., with regard to reproducibility, ISO 5725 requires validation in at least eight independent laboratories). 

The precision of an analytical method is a measure of the variability of repetitive measurements. Contributions from numerous sources affect precision, but the major components are within-laboratory (repeatability) and between-laboratory (reproducibility) variations. Precision is expressed as the relative standard deviation (or CV)... 

In summary, official German analytical methods for pesticide residues are always validated in several laboratories. These inter-laboratory studies avoid the acceptance of methods which cannot readily be reproduced in further laboratories and they do improve the ruggedness of analytical procedures applied. The recently introduced calibration with standards in matrix improves the trueness of the reported recovery data. Other aspects of validation (sample processing, analyte stability, extraction efficiency) are not considered. 

If analytical methods are validated in inter-laboratory validation studies, documentation should follow the requirements of the harmonized protocol of lUPAC. " However, multi-matrix/multi-residue methods are applicable to hundreds of pesticides in dozens of commodities and have to be validated at several concentration levels. Any complete documentation of validation results is impossible in that case. Some performance characteristics, e.g., the specificity of analyte detection, an appropriate calibration range and sufficient detection sensitivity, are prerequisites for the determination of acceptable trueness and precision and their publication is less important. The LOD and LOQ depend on special instmmentation, analysts involved, time, batches of chemicals, etc., and cannot easily be reproduced. Therefore, these characteristics are less important. A practical, frequently applied alternative is the publication only of trueness (most often in terms of recovery) and precision for each analyte at each level. No consensus seems to exist as to whether these analyte-parameter sets should be documented, e.g., separately for each commodity or accumulated for all experiments done with the same analyte. In the latter case, the applicability of methods with regard to commodities can be documented in separate tables without performance characteristics. 

Methods submitted include single- and multi-analyte methods for parent compounds and for degradates of concern. Pesticide regulatory methods are needed for each type of environmental matrix fate methods may be designed for soil, water, plant tissue, animal tissue or air, but are predominantly for soil and water. Analytical methods need to include a complete description of the procedure, materials and equipment in order to be completely reproducible. The methods should be practical and rapid and, to the extent possible while maintaining other quality objectives, inexpensive (often State and local regulatory agencies with few available resources need to utilize them). 

In general, the physical structure of the tissue must be broken down mechanically followed by an extraction procedure, before the sample can be analyzed. Homogenization using blenders, probe homogenizers, cell disrupters, sonicators, or pestle grinders is particularly useful for muscle, liver, and kidney samples. Regardless of the method used for tissue disruption, the pulse, volume of extraction solvent added, and temperature should be validated and standardized in order to ensure reproducible analytical results. During cell disruption, care should be taken to avoid heat build-up in the sample, because the analyte may be heat labile. 

A brief summary of EPA method requirements for tolerance enforcement methods is given in Table 1. Taken in total, these requirements ensure that the means to conduct the method are available to laboratories and that experimental evidence to establish method performance, on a substrate-by-substrate basis, is generated prior to analysis of samples and as part of each analytical set. Thus, an analyst who must generate data to support method performance in his or her hands can obtain whatever is required to reproduce the method. 

It is a regulatory requirement that analytical methods be developed to determine residues of concern in crops, feed, and food commodities as well as environmental samples (air, soil, and water). Methods for crops, feed, and food commodities are required for enforcement purposes but are also needed for a variety of other purposes, such as gathering monitoring data for risk assessment. For nearly any purpose, the methods must be robust, that is, when used by different analysts in several laboratories, they should provide reproducibly similar results. 

Method validation is needed to demonstrate the acceptability of the analytical method. A recovery test on a chemical being determined should be performed in order to verify the reliability of the series of analyses. Recovery studies are usually conducted by spiking untreated sediment with the target chemical at the deteetion limit, quantitation limit and in the range of 10-50 times the detection limit. The method is considered acceptable when the recoveries typically are greater than 70%. When the recovery is less than 70%, an improvement in the analytical methods is needed. Where this is not possible for technical reasons, then lower recovery levels may be acceptable provided that method validation has demonstrated that reproducible recoveries are obtained at a lower level of recovery. Analysis is usually done in duplicate or more, and the coefficient of variation (CV) should be less than 10% to ensure that recoveries will be consistently within the range 70-110%. 

For the spectrophotometric method, the evolved carbon disulfide is reacted with copper acetate and diethylamine to form a yellow copper complex which can be measured at 435 nm." The recoveries range between 70 and 90%. Reproducibility of this method was improved by reducing the time and the mode of sample pretreatment. Since all alkylenebis(dithiocarbamates) decompose to carbon disulfide by acid degradation, the above analytical methods are not selective. The result is the measured total residues of all alkylenebis(dithiocarbamates) related products. However, this method is recommended as standard method S15 for alkylenebis(dithiocarbamates) by the German Research Association. ... 

Analytical measurement Pros - Results obtained reflect well reality - Repeatability and reproducibility of results (at least between good qualified labs) - Measurements are independent of information/data sources - Multipurpose analytical methods can cover many compounds on a single run - Even the best model will ultimately need to be experimentally checked - Discovery of new emerging contaminants is possible... 

Both qualitative observations and quantitative measurements cannot be reproduced with absolute reliability. By reason of inevitable deviations, measured results vary within certain intervals and observations, mostly in form of decision tests, may fail. The reliability of analytical tests depends on the sample or the process to be controlled and the amount of the analyte, as well as on the analytical method applied and on the economical expenditure available. 

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