Sample dilution example

Example 6-2 The following standard addition plot was obtained for a competitive electrochemical enzyme immunoassay of the pesticide 2,4-D. A ground water sample (diluted 1 20 was subsequently assayed by the same protocol to yield a current signal of 65 nA. Calculate the concentration of 2,4-D in the original sample. 

The peak focusing allows injections of the extremely large sample volumes, which may easily reach 10% of the total volume of the LC LC columns. This is an important advantage considering the two-dimensional liquid chromatography. For example, the LC LC column effluent can be directly forwarded into an online SEC column for further separa-tion/characterization. The LC LC principle can be applied not only for polymer separations but also for reconcentration of polymer solutions, for example, of (diluted) effluents leaving other columns applied in polymer HPLC. This may be utilized in the multidimensional polymer HPLC. 

The analytical quality in such cases can be better assessed when CRMs of similar nature are available. For example, samples of arterial blood weighing 5 to 15 mg were withdrawn from different parts of a rabbit and were subjected to mineralization with hot 50% (v/v) HNO3 in a closed vessel microwave device. After adequate dilution end analysis of Ca, Mg and Fe was carried out by ICP/AES . 

Obviously, some procedures take less time than others. For example, sample concentration by the purge and trap technique that precedes VOC analyses takes only about 20 minutes. It is performed immediately prior to analysis on a multisample automated concentrator combined with an analytical instrument. The shortest of all sample preparation procedures is the waste dilution procedure, commonly known as dilute and shoot, which takes minutes. It consists of diluting a known volume of a concentrated waste sample in a known volume of a compatible solvent, followed by an injection into a gas chromatograph. 

The surrogate standard recoveries for some samples may be outside the control limits due to sample dilutions or matrix interferences. Such samples are usually clearly identified in the Case Narrative or in the laboratory reports, and their data are not qualified. For example, if a surrogate standard recovery is below 10 percent due to dilution, the result will not be rejected and will not be qualified as an estimated value. The chemist, however, may request from the laboratory the raw data for this sample in order to verify whether the dilution was justified and the interferences were truly present. 

Sometimes even valid and relevant data cannot be used for project decisions, particularly for projects that depend on a comparison of sample results to action levels. This may happen when samples are diluted during analysis. Although laboratory PQLs may be below the action levels, the RLs for the contaminants of concern in samples may be elevated due to dilutions and exceed the action levels. Example 5.11 demonstrates how the data may become unusable due to sample dilutions. 

The diligent analyst would develop a robust method with rigorous matrix effect tests on multiple lots, including hemolyzed and lipidemic samples. An initial test would be a spike-recovery evaluation on at least six individual lots. Samples should be spiked at or near the LLOQ, and at a high level near the ULOQ. If matrix interference were indicated by unacceptable relative error (RE) percentage in certain lots, the spiked sample of the unacceptable lots should be diluted with the standard calibrator matrix to estimate the minimum dilution requirement (MDR) at and above which the spike-recovery is acceptable. The spike-recovery test should then be repeated with the test samples diluted at the MDR. Note that this approach will increase the LLOQ for a less sensitive assay. If sensitivity is an issue, then other venues will be required to address the matrix effect problem. For example, the method can be modified to include sample clean-up, antibodies and/or assay conditions may be changed, or the study purpose may be tolerable to acknowledge that the method may not be selective for a few patients (whose data may require special interpretation). 

Chemical interference is practically non existent as a result of the high temperature of the plasma. On the other hand, physical interference may be observed. This stems from variations in the sample atomisation speed which is usually due to changes in nebulisation efficiency caused by differences in the physical properties of the solutions. Such effects may be caused by differences in viscosity or vapour tension between the sample solutions and the standards due, for example, to differences in acidity or total salt content. The technique most commonly used to correct this physical interference is the use of internal standards. In this technique a reference element is added at an identical concentration level to all the solutions under analysis, standards, blank and samples. For each element, the ratio of simultaneous measurements of the lines of the element and the internal standard is then determined in order to compensate for any deviation in the response of the plasma. If the internal standard behaves in the same way as the element to be determined, this method can be used to improve the reliability of the result by a factor of 2 to 5. It can also, however, introduce significant errors because not all elements behave in the same way. It is thus necessary to take care when using it. Alternatives to the internal standard method include incorporating the matrix into the standards and the blank, sample dilution, and the standard addition method. 

The Sample Dilution Factor relates the dilution from the reverse transcription reaction, based on a 1 pg RNA sample (equivalent to a 1 1 dilution) used directly in the PCR reaction. For example, a 1 8 dilution contains 125 ng input RNA, and the Sample Dilution Factor used in the equation is 8. A 1 2048 dilution contains... 

A further application for sample diluters arises when an initial extraction or dilution is required before the normal sequential operations of an AutoAnalyzer can be started. Examples of this can be considered in relation to the assay of serum cholesterol and blood glucose. For the AutoAnalyzer serum cholesterol method an initml extraction is required before the extract can be placed in the AutoAnalyzer cup, and difficulties can arise if an automatic diluter is used to perform this, since the extracting fluid precipitates the serum proteins at the interface in the diluter... 

Sample dilution is used to reduce the concentration of salts and endogenous materials in a sample matrix and is commonly applied to urine. Drug concentrations in urine are usually fairly high and allow this dilution without an adverse effect on sensitivity. While the amount of protein in a sample is usually a concern, protein concentrations in urine are negligible under normal physiological conditions. An example of a urine dilution procedure is reported for indinavir in which 1 mL urine was diluted with 650 qL acetonitrile so that... 

The extraction solvent should be chosen such that the analyte is extracted with as little co-extraction of interfering substances as possible. Furthermore, choose a solvent that is compatible with the solid-phase extraction method that is being used. For example, samples that are high in fat are best homogenized with nonpolar solvents, such as hexane. The use of hexane as an extractant dictates normal-phase sorbents with silica, alumina, Florisil, or a CN sorbent. Samples with high water content are best homogenized with acids, bases, or polar solvents, such as methanol, acetonitrile, or acetone. The use of methanol or one of the water-miscible solvents suggests the use of reversed phase (C-18) and dilution of the extract with water or buffer. Thus, the choice of the extraction solvent will dictate the type of sorbent that will be used in SPE. 

Let us insist on one of the main interests of the semi-deterministic deconvolution procedure. The measuring range can vary from 0.5 mg NOJ/L to several g NOg/L with the same pathlength (10 mm, for example) without dilution. For the purpose, as the wavelengths window used for the spectrum exploitation has to be adapted to the expected concentration, the precision varies in consequence and can be rather coarse for the intermediate range of concentration. This is the reason why the sample dilution is a good compromise in order to be able to apply the semi-deterministic method, the characteristics of which are presented in Table 1. 

Sample dilution is often required to match the expected analyte concentration with the dynamic concentration range of the analytical procedure. Clinical analyses, where several parameters are routinely determined in large sample batches, is a good example of this requirement. Extensive dilution of biological fluids is common practice in view of the high analyte concentration and the need to minimise matrix effects. However, sample dilution is usually time-consuming, can degrade precision and lead to systematic errors. 

Our laboratory has been using an Avagotech reader for evaluation of reagents for HIV and cotinine lateral flow assays. Figure 2. shows an example of results for a sample diluted in our prototype HIV antibody detection lateral flow test strips. The Avagotech reader allows the data to be plotted and coefficients of variation to be determined, making the screening more quantitative and less subjective. 

If interference occurs, the impact may be evaluated by assessing the amount of antigen required to reduce the antibody signal (positive) to background for a range of antibody concentrations. Other approaches, for example, acid dissociation of immune complexes [38], removal of theexcess biological by solid-phase adsorption, and/oruse of an assay that allows sufficient sample dilution to avoid this problem, may be considered. Such approaches need to be validated for effectiveness and adopted on a case-by-case basis. 

FIGURE 11.1 As is workflow example diagram for manual sample dilution. 

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