Within laboratory precision

Verification implies that the laboratory investigates trueness and precision in particular. Elements which should be included in a full validation of an analytical method are specificity, calibration curve, precision between laboratories and/or precision within laboratories, trueness, measuring range, LOD, LOQ, robustness and sensitivity. The numbers of analyses required by the NMKL standard and the criteria for the adoption of quantitative methods are summarized in Table 10. 

Precision is the degree of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample under prescribed conditions. Precision may be considered at three levels repeatability (precision under the same operating conditions over a short interval of time), intermediate precision (precision within-laboratory variations different days, different analysis, different equipment, etc.), and reproducibility (precision between laboratories, collaborative studies, usually applied to standardization of methodology). 

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)... 

Method performance study All laboratories follow the same written protocol and use the same test method to measure a quantity (usually concentration of an analyte) in sets of identical test samples. The results are used to estimate the performance characteristics of the method, which are usually within-laboratory- and between-laboratory precision and - if relevant - additional parameters such as sensitivity, limit of detection, recovery, and internal quality control parameters (IUPAC Orange Book [1997, 2000]). 

We will address aspects of reproducibility, which has previously been defined as, the precision between laboratories . It has also been defined as total between-laboratory precision . This is a measure of the ability of different laboratories to evaluate each other. Reproducibility includes all the measurement errors or variances, including the within-laboratory error. Other terms include precision, defined as the closeness of agreement between independent test results obtained under stipulated conditions [3] and repeatability, or the precision for the same analyst within the same laboratory, or within-laboratory precision . Note that for none of these definitions do we require the true value for an analytical sample . In practice we do not know the true analyte value unless we have created the sample, and then it is only known to a given certainty (i.e., within a determined uncertainty). 

If repeatability is the only estimate of precision that is obtained, this is unlikely to be representative of the variability observed when the method is used over a long period of time. Intermediate precision is often more relevant - this expresses the within-laboratory variation or within-laboratory reproducibility (different days, different analysts, different equipment, etc.). This is initially obtained from validation studies and confirmed later by examining the results obtained for quality control material measured over a period of about three months (see the quality control (QC) charts in Chapter 6). 

The values quoted in Table 4.3 refer to the spread of results expected when a given sample is analysed in a number of separate laboratories. For repeat analyses carried out by one operator in a single laboratory, the coefficient of variation (%CV) would typically be one half to two thirds of the values shown in Table 4.3. For within-laboratory reproducibility (intermediate precision), the %CV should not be greater than the reproducibility %CV for the given concentration in Table 4.3. 

Within-laboratory reproducibility studies should cover a period of three or more months and these data may need to be collected during the routine use of the method. It is possible, however, to estimate the intermediate precision more rapidly by deliberately changing the analyst, instrument, etc. and carrying out an analysis of variance (ANOVA) [9]. Different operators using different instruments, where these variations occur during the routine use of the method, should generate the data. 

Within-laboratory reproducibility/intermediate precision Precision under conditions where independent test results are obtained with the same method on identical test items in the same laboratory by different operators using different equipment on different days. 

The uncertainty of a measurement is always a very important consideration but it is especially so with durability tests. In addition to the basic physical test methods, there are the complications of exposure conditions, tests spanning long times and the process of extrapolating results to make predictions. The combination of these factors will inevitably lead to large uncertainties that can very easily be of such a magnitude that any conclusions are meaningless. Terms used to describe precision include repeatability, which refers to within laboratory variation and reproducibility, which refers to variation between laboratories. 

Intralaboratory or within-laboratory precision refers to the precision of a test method when the results are obtained by the same operator in the same laboratory using the same apparatus. In some cases, the precision is applied to data gathered by a different operator in the same laboratory using the same apparatus. Thus, intralaboratory precision has an expanded meaning insofar as it can be applied to laboratory precision. 

Intermediate precision (day-to-day Precision including within-laboratory variations, e.g., different... 

Intermediate precision includes within laboratories variations different days, different analysts, different equipment, etc. 

In a protocol about collaborative studies [10] it is also considered what is called preliminary estimates of precision. Among these the protocol defines the total within-laboratory standard deviation . This includes both the within-run or intra-assay variation (= repeatability) and the between-run or inter-assay variation. The latter means that one has measured on different days and preferably has used different calibration curves. It can be considered as a within-laboratory reproducibility. These estimates can be determined prior to an interlaboratory method performance study. The total within-laboratory standard deviation may be estimated fi-om ruggedness trials [10]. 

Within-laboratory uncertainty is derived from intermediate precision and includes only the repeatability error and the run error. 

Intermediate Precision. Intermediate precision expresses within-laboratory variation and is generally performed on different days using different analysts, equipment, and sample preparations. This test may not be applicable if the laboratory has only one workstation. Additionally, this test may not be appropriate for automated workstations that are operating under the same environment and controls within a laboratory. This assumption is made on the basis that the automated workstations are identical (i.e., same configuration, same software and hardware) and that they have been suitably qualified and maintained to a consistent standard and operate under a similar climatic environment. The influence of the analyst is reduced to the preparation of solvents, and this should be covered by the robustness studies. 

The ideal validated method would be the one that has progressed fully through a collaborative study in accordance with international protocols for the design, conduct, and interpretation of method performance studies. A typical study of a determinative method conducted in accordance with the internationally harmonized International Organization for Standardization (ISO)/International Union for Pure and Applied Chemistry (IUPAC)/AOAC International (AOAC) protocol would require a minimum of up to five test materials including blind replicates or split-level samples to assess within-laboratory repeatability parameters, and eight participating laboratories (15). Included with the intended use should be recommended performance criteria for accuracy, precision and recovery. 

Precision should be measured using a minimum of five determinations per concentration. A minimum of three concentrations in the range of expected concentrations is recommended. The precision determined at each concentration level should not exceed 15 % of the coefficient of variation (CV) except for the LLOQ, where it should not exceed 20 % of the CV. Precision is further subdivided into within-run, intrabatch precision or repeatability, which assesses precision during a single analytical run, and between-run, interbatch precision or reproducibility, which measures precision with time, and may involve different analysts, equipment, reagents, and laboratories. Since it is not always easy to obtain data about the reproducibility in the strict sense it often makes sense to use intermediate precision this expresses within-laboratories variations on different days, by different analysts, and on different equipment, etc. [60],... 

Intermediate precision6 Intermediate precision expresses within-laboratories variations different days, different analysts, different equipment, etc. This characteristic may be assessed on the worst-case surface(s) representing poor recovery and/or high variability +... 

The parameters describing the scattering of a test result under repeatability and reproducibility conditions are the corresponding standard deviations. The repeatability limit, "r , is the within-laboratory precision and describes the maximum expected value of the difference between two individual test results obtained under repeatabil-... 

Within laboratory (repeatability conditions) precision according to ISO 5725... 

As a result of the pre-validation work, which included a within-laboratory precision experiment carried out in two different laboratories at concentrations of 2.1 mg HMDA and 12.1 mg EDA per kg food simulant, the performance characteristics in Table 10-7 were obtained at the 95 % probability level. 

The precision has been defined in the ISO 5725-1 standard as the closeness of agreement between independent test results obtained under stipulated conditions [28]. Many factors may contribute to the variability of test results obtained with the same method on identical samples, including (but not limited to) the operators), the equipment, the reagents, the RM(s), the environment, the time between measurements, and the laboratories. The maximal variability of test results is explained by the reproducibility (R) of the method. All factors that have influence on the variability of a test method should be taken into consideration when assessing the reproducibility. The repeatability (r) is assessed by keeping all the above-mentioned factors constant (e.g., same operator, same equipment, same laboratory, short time interval). It is a measure of the minimum variability of a method. The intermediate precision is situated between the two extreme measures of precision repeatability and reproducibility. The terms within-laboratory reproducibility (w), long-term precision, and so on, are often used to demonstrate the intermediate precision of a method. For a correct interpretation of the intermediate precision, the factors that have been taken into account should be known. 

Example. The Repeatability and Within-Laboratory Reproducibility of the Analysis of Cd with ET-AAS is Assessed by Means of a Precision Experiment A blank sample has been spiked at a level of 75 ng g-1 Cd. The samples have been analyzed in duplicate on six different analytical runs according to the prescribed analytical procedure (SOP 22/F/1701) [37], The results are reported in Table 6.4. 

The repeatability and within-laboratory reproducibility (expressed as CV percent) are evaluated against the Horwitz equation according to the EU Commission Decision [4]. The Horwitz equation is an empirical relationship between the concentration of the analyte and the precision of the method. Initially the Horwitz equation was developed from data obtained during collaborative trials [38, 39]. The following equation for the maximum reproducibility CV is valid. The maximum repeatability is between one-half and two-thirds of the CVR (percent). 

Intermediate precision is the within laboratory variation over a long period of time. The standard deviation will be intermediate in value between that obtained under repeatability and that obtained under reproducibility conditions for similar samples using the same method. 

Azen 1979 Massart et al. 1990 Gardiner 1997). This random effects model is often used in analytical chemistry to breakdown a total precision into its components such as between-days and within-days, or between-laboratories and within-laboratories in collaborative trials, to validate an analytical method using reference material. 

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