Reproducibility within laboratory

If we don t have such an ideal control sample, but only one with a matrix different from the routine sample (e.g. a standard solution) than we have to consider also the uncertainty component arising from changes in the matrix. For this purpose we use the (repeatability) standard deviation calculated from repeated measurements of our routine samples (performed e.g. for a range control chart). When we estimate the reproducibility within laboratory we now have to combine both contributions by calculating the square root of the sum of squares. 

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 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)... 

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. 

Method validation provides information concerning the method s performance capabilities and limitations, when applied under routine circumstances and when it is within statistical control, and can be used to set the QC limits. The warning and action limits are commonly set at twice and three times the within-laboratory reproducibility, respectively. When the method is used on a regular basis, periodic measurement of QC samples and the plotting of these data on QC charts is required to ensure that the method is still within statistical control. The frequency of QC checks should not normally be set at less than 5% of the sample throughput. When the method is new, it may be set much higher. Quality control charts are discussed in Chapter 6. 

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. 

There is a recent trend towards simultaneous CE separations of several classes of food additives. This has so far been applied to soft drinks and preserved fruits, but could also be used for other food products. An MEKC method was published (Lin et al., 2000) for simultaneous separation of intense sweeteners (dulcin, aspartame, saccharin and acesulfame K) and some preservatives (sorbic and benzoic acids, sodium dehydroacetate, methyl-, ethyl-, propyl- and isopropyl- p-hydroxybenzoates) in preserved fruits. Ion pair extraction and SPE cleanup were used prior to CE analysis. The average recovery of these various additives was 90% with good within-laboratory reproducibility of results. Another procedure was described by Frazier et al. (2000b) for separation of intense sweeteners, preservatives and colours as well as caffeine and caramel in soft drinks. Using the MEKC mode, separation was obtained in 15 min. The aqueous phase was 20 mM carbonate buffer at pH 9.5 and the micellar phase was 62 mM sodium dodecyl sulphate. A diode array detector was used for quantification in the range 190-600 nm, and limits of quantification of 0.01 mg/1 per analyte were reported. The authors observed that their procedure requires further validation for quantitative analysis. 

Quantification of the within laboratory reproducibility (or better the imprecision)... 

We are starting with the case where we have a control sample that covers the whole analytical process inclnding all sample preparation steps. The matrix of the control sample is similar to that of the routine samples. Then the standard deviation of the analysis of this sample (under between-batch conditions) can be used directly as an estimate for the reproducibility within the laboratory. The standard deviation can be taken directly from a control chart for this control sample (see chapterl3). In the table two examples are shown for different concentration levels. 

Reproducibility within the Laboratory R Control Sample Covering the Whole Analytical Process... 

The within-laboratory reproducibility at that concentration level can simply be estimated from the analyses of the control sample... 

Although DC maturation can be used to detect sensitizing capacity, major concerns remain on this assay (i) the limited reproducibility within and between laboratories due to inter-donor variability and variations in cell isolation and culture techniques (ii) the lack of sensitivity and dynamic range [122]. To circumvent interdonor variability cell lines such as THP-1, U937, KG-1 and MUTZ-3 have been used. 

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]. 

If there is any doubt about whether 100% of the analyte is presented to the measuring system or that the response of the calibrated system leads to no bias, then the assumptions must be tested during method validation and appropriate actions taken. If a series of measurements of a CRM (not used for calibration) leads to the conclusion that there is significant bias in the observed measurement result, the result should be corrected, and the measurement uncertainty should include the uncertainty of the measurement of the bias. If the bias is considered insignificant, no correction need be made, but measuring the bias and concluding that it is zero adds uncertainty (perhaps the bias was not really zero but is less than the uncertainty of its measurement). One approach to measurement uncertainty is therefore to include CRMs in the batch to be used to correct for bias, and then the uncertainty of estimation of bias, which includes the uncertainty of the quantity value of the CRM, is combined with the within-laboratory reproducibility. In some fields of analysis it is held that routine measurement and correction for bias... 

The expected maximum difference between two results obtained by repeated application of the analytical procedure to an identical test sample under different conditions but in the same laboratory. The measure for the within-laboratory reproducibility (Aw) is the standard deviation (-SaJ-... 

For series of measurements of sufficient size (usually not less than 6), the within-laboratory reproducibility is defined as... 

Within-laboratory reproducibility should be obtained by one or several operators with the same equipment in the same laboratory at different days using the same method. 

Repeatability Within-laboratory reproducibility Between-laboratory reproducibility Same L, T, A, I Same L different T I and A may be different Different L, T, A, I... 

If it is not possible to involve additional laboratories for the determination of the between-laboratory reproducibility, then the within-laboratory reproducibility may be used to get an estimate of the between-laboratory reproducibility. The reproducibility of the method may be dependent upon the mass fraction of the analyte in the test sample. It is therefore recommended, when studying the reproducibility, to investigate whether a relation exists between concentration and reproducibility. The measurement series should be greater than 8. 

TEMPERATURE TRANSFER STANDARD. A device for the transfer of a temperature scale from one standardizing laboratory to another. One form consists of a sample of a purified material, the freezing point of which (when realized by a prescribed technique) is reproducible within narrow limits. Materials commonly employed are metals, such as zinc and tin, and organic compounds, such as benzoic acid, phenol, naphthalene, and phthalic anhydride. Another form is a tungsten ribbon-filament lamp, characterized by a stable lamp current-brightness temperature relation. This device is particularly useful for temperatures above 1.050WC. 

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],... 

Since the historical PV weak force origin /3-decay experiment of 60Co [ 106], theoreticians presumed that the tiny parity violating WNC at molecular and subatomic levels may also allow a distinction between mirror image molecules at the macroscopic level as well. This is because PV-WNC at the molecular level may be a candidate for the homochiral scenario under terrestrial and extraterrestrial conditions [1,2,104,109-118]. The WNC, however, did not induce any observable PV effects between enantiomers in their ground states because of the minuscule PV energy difference (PVED) of 10 19 eV and/or negligibly small 10 - % ee in racemates. Theoreticians also proposed several possible amplification mechanisms at reproducible detection levels within laboratory time scales and at terrestrial locations [113,117,118]. 

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