Sample preparation, generally method validation

Before a new analytical method or sample preparation technique is to be implemented, it must be validated. The various figures of merit need to be determined during the validation process. Random and systematic errors are measured in terms of precision and bias. The detection limit is established for each analyte. The accuracy and precision are determined at the concentration range where the method is to be used. The linear dynamic range is established and the calibration sensitivity is measured. In general, method validation provides a comprehensive picture of the merits of a new method and provides a basis for comparison with existing methods. 

Validation of methods for quantitative determination of impurities includes precision studies. Repeatability is generally assessed by analysis of the same sample or samples prepared by the same analyst in replicate assays within a short duration of time. Repeatability should be assessed using (i) a minimum of nine determinations covering the specified range for the procedure (e.g., three concentrations/three replicates each) or (ii) a minimum of six determinations at 100% of the test concentration. Repeatability is evaluated by averaging the mean results from replicate assays and calculating the standard deviation (SD) and RSD. Repeatability of the method can be stated as either SD or RSD values. If an instrument is required for assay performance, then the same instrument should be used for the replicate assays. 

Validation is needed to demonstrate that the analytical method complies with established criteria for different performance characteristics [82]. When these different characteristics are being evaluated individually, this is generally done for the analytical method as such—where the input is the purified or isolated analyte and the output is the analytical result. However, MU covers the whole analytical procedure, starting from the original sample lot. The assessment of MU (see Section 8.2.2) is in line with the so-called modular validation approach. Modular validation refers to the modularity of an analytical procedure divided up into several sequential steps needed to analyze the material. These may be sample preparation, analyte extraction, and analyte determination (Figure 7). Each step in the procedure can be seen as an analytical system and can thus be validated separately and combined... 

General Limitations of Urine Enzyme Analysis The correct sample handling methodologies have not been well validated for all enzymes in all species and it may require some effort on the part of the investigator to determine the need and optimal method for sample preparation. 

This book intends to supply the basic information necessary to apply the methods of vibrational spectroscopy, to design experimental procedures, to perform and evaluate experiments. It does not intend to provide a market survey of the instruments which are available at present, because such information would very soon be outdated. However, the general principles of the instruments and their accessories, which remain valid, are discussed. Details concerning sample preparation and the recording of the spectra, which is the subject of introductory courses, are assumed to be known. Special procedures which are described in monographs, such as Fourier transformation or chemometric methods, are also not exhaustively described. This book has been written for graduate students as well as for experienced scientists who intend to update their knowledge. 

One of the main constraints on conventional procedures for sample preparation is their lack of uniformity. In fact, many users introduce specific modifications in the general procedures that eventually result in enormous differences in the way they are implemented. Such a lack of uniformity precludes comparison of results obtained using what was seemingly the same procedure and validation of the ensuing methods. Comparability of the results can thus only be achieved by using actually identical procedures this justifies the efforts currently in progress at standardizing sample preparation procedures. 

Unless care is exercised by the analyst, both these forms of contamination can go unnoticed and erroneous results may be reported for individual samples. Problems with cross-contamination should normally be identified during the validation phase of method development by the judicious use of blanks to test for problems with general laboratory contamination, sample preparation, and the autosampler. Carryover is... 

In conjunction with separation optimization, the use of automated sample preparation methods, to increase throughput, should be investigated for late-stage methods. In general, it is preferable to develop the automated sample preparation procedure prior to validation of the method, to incorporate validation considerations of the automation into the validation scheme. 

Preparation of Standards and Curves In the absence of a true blank control matrix, standards can be prepared in a protein buffer at multiple (generally 9 11) concentrations for the initial test of assay range. For method validation and sample assay, 6 8 nonzero concentrations of standards plus anchor points should be used to define a curvilinear standard curve. If a commercial kit is to be used, it is preferred that the standards be prepared from a bulk reference material to assure the consistencies of the standard as well as adding enough standard points to properly define the regression model. Before replacing the kit standards with those prepared... 

The results obtained from the investigation of the crystallochemical properties of a family of mixed Li-vanadates of general formula LiCoyNi(i.y)V04 are reported. This kind of material is believed to be a valid alternative to the traditional positive electrode materials in the Li-ion cell, an electrochemical power source of outstanding importance in the portable electronics field. The powders, prepared via two different methods (a wet chemistry route and a solid state method), have been characterised by XRPD analysis, NMR and diffuse reflectance Vis-NIR spectroscopy. The findings allowed to correlate the electrochemical performance of the vanadates, which is higher for the samples prepared via wet chemistry, to the crystallinity of the powders and to the transition metal cations distribution. 

General sample preparation will be discussed in this chapter, but instmment-specihc sample preparation is included in the appropriate chapter on each technique. Method validation and documentation will not be covered as the focus of this text is on instrumentation. The text by Christian cited in the bibliography has an excellent introduction to validation and documentation for the interested student. 

For method validation studies, QCs are often prepared and analyzed fresh on the same day, but when QCs are being prepared and then stored for subsequent use during sample analysis it is advisable to run some type of QC check of the prepared batch of QCs prior to use. Generally, QC replicates are prepared and assayed along with a standard curve and a set of blanks. The number of replicates used will depend of the availability of control matrix (and thus the number of available QCs), laboratory SOPs and/or historical information about the method with respect to previous batches of QCs made. 

In the next step in the preparation of a validation, three (or preferably more) samples with the envisaged lowest concentration after sample preparation considering the estimated recovery rate should be analyzed. If it is at all possible, an internal standard should be added to the samples. A final decision as to whether to use a final method with or without internal standardization can then be taken later. An internal standard is generally the best quality control possibility. Each analysis sample will then be individually controlled with virtually no additional effort. 

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