Reproducibility of an analytical

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. 

Reproducibility as defined by ICH [2,3] represents the precision obtained between laboratories (Table 5). The objective is to verify that the method will provide the same results in different laboratories. The reproducibility of an analytical method is determined by analyzing aliquots from homogeneous lots in... 

Figure 6.5. Experimental design for the assessment of the repeatability, intermediate precision and the reproducibility of an analytical method.

Reproducibility, as defined by ICH, represents the precision obtained between laboratories with the objective of verifying if the method will provide the same results in different laboratories. The reproducibility of an analytical method is determined by analyzing aliquots from homogeneous lots in different laboratories with different analysts, and by using operational and environmental conditions that may differ from, but are still within the specified, parameters of the method (interlaboratory tests). Various parameters affect reproducibility. These include differences in room environment (temperature and humidity), operators with different experience, equipment with different characteristics (e.g., delay volume of an HPLC system), variations in material and instrument conditions (e.g., in HPLC), mobile phases composition, pH, flow rate of mobile phase, columns from different suppliers or different batches, solvents, reagents, and other material with different quality. 

It is well understood that the majority of variation in metabonomic experiments comes from the biological rather than analytical variation [83], Nevertheless, it is critical that the reproducibility of an analytical method is acceptable and that... 

The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Precision may be considered at three levels repeatability (within run) intermediate precision (over time) and reproducibility (inter-laboratory). 

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 ... 

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)... 

Capillary electrophoresis offers several useful methods for (i) fast, highly efficient separations of ionic species (ii) fast separations of macromolecules (biopolymers) and (iii) development of small volume separations-based sensors. The very low-solvent flow (l-10nL min-1) CE technique, which is capable of providing exceptional separation efficiencies, places great demands on injection, detection and the other processes involved. The total volume of the capillaries typically used in CE is a few microlitres. CE instrumentation must deliver nL volumes reproducibly every time. The peak width of an analyte obtained from an electropherogram depends not only on the bandwidth of the analyte in the capillary but also on the migration rate of the analyte. 

According to USP 28 [1], validation of an analytical method is the process by which it is established, through the conduct of laboratory studies, that the performance characteristics of the method meet the requirements for the intended analytical applications. Therefore, validation is an important step in determining the reliability and reproducibility of the method because it is able to confirm that the intended method is suitable to be conducted on a particular system. 

The robustness of an analytical method can be defined as a measure of the capability of the method to remain unaffected by small, but deliberate, variations in method parameters. The parameter therefore provides an indication of the method reliability during normal usage. The ruggedness of a method is the degree of reproducibility of test results obtained by the analysis of the same samples under a variety of conditions, such as different laboratories, different analysts, different instruments, different lot of reagents, different days, etc. 

The management of an analytical chemistry laboratory involves a number of different but related operations. Analysts will be concerned with the development and routine application of analytical methods under optimum conditions. Instruments have to be set up to operate efficiently, reproducibly and reliably, sometimes over long periods and for a variety of analyses. Results will need to be recorded and presented so that the maximum information may be extracted from them. Repetitive analysis under identical conditions is often required, for instance, in quality assurance programmes. Hence a large number of results will need to be collated and interpreted so that conclusions may be drawn from their overall pattern. The progress of samples through a laboratory needs to be logged and results presented, stored, transmitted and retrieved in an ordered manner. Computers and microprocessors can contribute to these operations in a variety of ways. 

In order to evaluate pump flow rate reproducibility and pulsation, one method is commonly used to assess gradient formation capability. A certain amount of an analyte with adequate molar absorptivity at the wavelength employed for detection is introduced into one of the mobile phases employed to create the gradient. In the case described, 5% acetone was introduced into the mobile phase, distributed to the system by pump B. No UV-absorbing analyte was introduced into mobile phase A. The fractional flow rate of pump B relative to the total flow rate of the system (mandated by the sum of the flow rates of pumps A and B) was increased in individual steps to account for 0, 3,6,12.5,25, 50, and 100% fractional rates. The total flow for the system was maintained at 300 /jL/ min (for 24 columns), resulting in a per column flow rate of 12.5 /iL/min/column. 

The first option involves the obtainement of an analytic function that reproduces the interaction energy between couples of molecules which has been calculated by solving the Schrodinger equation usually by means of an ab initio method. The advantge of this possibility is that information about any potential energy hypersurface point can be obtained from the calculation whereas experimentally this is not always possible. The practical procedure in order to build up an ab initio pair potential for the interaction between two molecules a and P can be divided in four steps. 

The El source has been the most widely used ion source over the past 60 years and continues to be the method of choice for the analysis (either qualitative or quantitative) of small- to medium-sized volatile organic compounds. The inherent reproducibility of the mass spectra has enabled the assembly of large spectral libraries. Computers associated with current generation instruments can efficiently (in a few seconds) search an unknown mass spectrum against tens of thousands of reference spectra in order to aid in the identification of an analyte. The general scheme of an El source includes the introduction of the vaporized analyte molecules into the ionization chamber, exposure of those molecules... 

In the context of an analytical method to establish the accuracy, precision, reproducibility, response function and the specificity of the analytical method with reference to the biological matrix to be examined and the analyte to be quantified. 

The precision of a test method is the variability between test results obtained on the same material using a specific test method (ASTM, 2004 Patnaik, 2004). The precision of a test is usually unrelated to its accuracy. The results may be precise, but not necessarily accurate. In fact, the precision of an analytical method is the amount of scatter in the results obtained from multiple analyses of a homogeneous sample. To be meaningful, the precision study must be performed using the exact sample and standard preparation procedures that will be used in the final method. Precision is expressed as repeatability and reproducibility. 

Kovats indices (/-values) are based on the retention time of an analyte compared to retention times of the series of n-alkanes. For a particular GC phase, /-values are very reproducible from one column or from one GC to another. However, they are slightly affected by GC programming conditions. n-Alkanes have most affinity for non-polar phases and tend to elute more quickly from polar phases. In contrast, a polar analyte will elute more slowly from a polar phase and thus relative to the n-alkanes, its retention time and thus its /-value will increase as the polarity of the... 

The ruggedness of an analytical method can generally be described as the ability to reproduce an analytical method in different laboratories or in different circumstances without the occurrence of unexpected differences in the obtained results. 

The precision of an analytical method is usually expressed as the standard deviation or relative standard deviation (coefficient of variation) of a series of measurements. Precision represents repeatability or reproducibility of the analytical method under normal operating conditions. Precision determinations permit an estimate of the reliability of single determinations and are commonly in the range of 0.3 to 3% for dosage form assays. 

The precision of an analytical method can be defined as the pattern of variation of single assays on a uniform sample. The precision serves to identify random errors and is described by the repeatability (variability within a laboratory) and reproducibility (variation between different laboratories). 

The ruggedness of an analytical method is determined by analysis of aliquots from homogeneous samples in different laboratories, by different analysts, using operational and environmental conditions that may differ but are still within the specihed parameters of the assay. The degree of reproducibility of test results is then determined as a function of the assay variables. This reproducibility may be compared to the precision of the assay under normal conditions to obtain a measure of the ruggedness of the analytical method. 

Reproducibility Reproducibility measures the precision between laboratories. This parameter is considered in the standardization of an analytical procedure (e.g., inclusion of procedures in pharmacopeias and method transfer between different laboratories). 

Quantification range the range of concentration, including ULOQ and LLOQ, that can be quantified reliably and reproducibly with accuracy and precision through the use of a concentration-response relationship. Recovery the extraction efficiency of an analytical process, reported as a percentage of the known amount of an analyte carried through the sample extraction and processing steps of the method. 

Recovery of the analyte need not be 100%, but the extent of recovery of an analyte and of the internal standard should be consistent, precise, and reproducible. Recovery experiments should be performed by comparing the analytical results for extracted samples at three concentrations (low, medium, and high) with unextracted standards that represent 100% recovery. 

Recovery/Extraction Efficiency A ratio between the response of an analyte spiked into a blank matrix preextraction and the response of the same analyte spiked into a blank matrix postextraction. Although the recovery of an analyte need not be close to 100%, the extent of the recovery at all QC levels should be consistent, precise, and reproducible. 

The detectors used with UPLC systems have to be able to handle very fast scanning methods because peak half-height widths of around 1 s are typically obtained with columns packed with 1.7-p.m particles. In order to accurately and reproducibly integrate an analyte peak, the detector sampling rate must be high enough to capture enough data points across the peak. Conceptually, the sensitivity increase for UPLC... 

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