Spiked material

Matrix RMs - spiked materials, lake water fortified with trace elements and milk powder spiked with organic contaminants. 

It is required for quantitative purity assays, and it must be established across the specified range of the analytical procedure. This can be done, by establishing the recovery rate over the range of the method. Alternatively, a method comparison between a validated method and a new method can be performed. Accuracy can be determined by spiking degraded, aggregated, pure or impure material into a known amount of sample. A theoretical recovery would then be calculated and the spike material analyzed using the chosen method. The actual recovery should then be compared to the theoretical recovery to calculate the accuracy of the method. Accuracy in this case would be reported as percent recovery. 

Spiked materials These are similar to fortified materials, indeed to some extent the terms are interchangeable. Spiking does not necessarily have to be restricted to the airalyte of interest. 

The advantage of IDMS in comparison to other quantification strategies (external calibration or standard addition) is that analyte recovery does not have to be quantitative, providing that isotopic equilibrium has been achieved between all of the analyte and the added spike material. High accuracy analysis by IDMS is now well established.53 In the following paragraphs selected examples of IDMS use will be discussed briefly. 

Matrix effects play an important role in the accuracy and precision of a measurement. Sample preparation steps are often sensitive to the matrix. Matrix spikes are used to determine their effect on sample preparation and analysis. Matrix spiking is done by adding a known quantity of a component that is similar to the analyte but not present in the sample originally. The sample is then analyzed for the presence of the spiked material to evaluate the matrix effects. It is important to be certain that the extraction recovers most of the analytes, and spike recovery is usually required to be at least 70%. The matrix spike can be used to accept or reject a method. 

Fig. 10 Effect of formulation on virus reduction and product recovery. Protein G was formulated in buffer containing albumin (0% or 2%) and NaCI (OmM or 150mM). Virus was added and a sample was immediately removed for virus titration (Pre-FD). The virus-spiked material was aseptically filled into vials, freeze dried, and then heated at 80°C, 72 hr. Mock-spiked Protein G was processed like the virus-spiked samples but was used to measure product recovery (potency).

So, the methods characteristic of each test, comprising taken together a type of tests, must undergo validation testing of their results. This is the implementation of the method, and the establishment of a standard for its performance. For the standardization of quantitative methods, this consists at a minimum of a determination of trueness when blank utilization, certified reference materials (or reference materials, or spiking materials) or collaborative trials are used, repeatability (r) with repetition,... 

At the same time, internal quality control must be carried out to verify the performance stability of the limited-scope performance of the method. Triply redundant verification methods are carried out with regard to control of first, second and/or third line, each set of methods applied to a particular type of test. The first line of verification involves pro forma repetition of all the steps of the test, in order to establish repeatability or reproducibility for quantitative tests, and to verify the range of sensitivity or detection for qualitative tests. This verification is performed by the experimenter himself, as part of the proper performance of the test. A second line of verification is put into operation by administrative decision, and includes testing with blind samples, repetition of samples, internal audit procedures, etc. If necessary, a third line of verification can be set up by the use of certified reference materials (or spiking materials), or through collaborative trials. These procedures are based on external cooperation. External audit procedures and complaints handling procedures are also part of this third line of verification. 

Samples must be homogeneous and tested for homogeneity, and should be coded at random, including the two or more blind replicates. A blank or negative control, and, if available, reference materials should be provided. Spiked materials are recommended for recovery study, incurred materials for residues study. 

As shown in section 2.3 above, the method development and validation necessitate the preparation of several working materials to be able to verify each step of the method. Raw or cleaned-up extracts, spiked or not. spiked natural or artificial materials are all tools to which the operator will refer. In the method validation they serve mainly to establish the uncertainty of individual steps but also to verify the absence of losses of substance (spiked materials) or contamination (spiked materials and blanks). They are... 

The method performance study focuses on the analytical method. In order to achieve a constructive work the organiser should assure that the method to be used by the participants has been investigated in detail prior to the start of the interlaboratory study. This preliminary investigation should be undertaken by a senior analyst. It should lead to a detailed draft analytical protocol, to a clear definition of the type and number of samples to be distributed and analysed, to indications on the repeatability of the method and its long-term reproducibility. In parallel, a study on the production of proper samples should be undertaken. Additional materials can also be prepared, e.g. calibration tools (pure calibrants or solutions), or blanks with a similar matrix than the test sample, spiked materials, etc. 

Recovery describes the ability of an analytical system response to quantitate an amount of analyte that has been spiked into sample matrix. It is usually conducted using several samples or pools and the spiked material may be either calibrator, QC, or another source of the biomarker to be measured. 

The known amount Ny added is called a "spike". Rx and Ry can be defined as the isotope ratios R of the unknown sample X and of the "spike" respectively. Note that in this example - since the unknown sample is monoisotopic (containing l only) - flx = and that Ry = 0 if a pure I isotope is used as spike material. In fact an isotope is seldom, if ever, 100% pure. Consequently it is better to use the detection limit for the... 

It is seen that the ratio of an unknown number of atoms Wx to a known number Ny of atoms ("spike") is again solely a function of isotope ratios. However, also sample and spike have to be measured for their isotopic composition (measurement of Rx and Ry) in addition to the blend (measurement of Rb). This can be performed on separate samples of unknown and spike" material and does not require quantitativity in sample-taking. It is trivial to note that the same reference isotope must be used in sample, spike and blend ratios. However, any isotope can be used as reference isotope. 

Spike material has to be an isotopically enriched material, is hence available to limited amounts only and/or can be expensive. Reverse isotope dilution can be used to perform its assay because it can be performed on minute amounts or very small concentrations (ug.mr ). 

Furthermore, complete or precise isotope analysis of the spike material is not essen-... 

Very enriched or depleted spike material is not always needed generally a variety of parameter combinations allows to reach an uncertainty close to the theoretical optimum which is for the given ratio measurement technique. 

This method of calibration has the disadvantage that drift in instrumental response may cause significant errors because the calibration and sample measurements are made some time apart. It has been widely used for organic IDMS but is less attractive for inorganic IDMS where several isotopes may be present at significant levels in both the sample and the spike material. In such instances the calculation of calibration data will be more difficult. This is because both the natural and the spike materials will often contain both isotopes of interest for the IDMS measurement. This in turn leads to a non-linear relationship between the signals observed and the amounts used to create a blend of the natural and spike materials. 

There is a theoretical optimum for spiking samples to achieve the best precision for the ratio measurements. The error propagated by this measurement factor alters as one moves away from the theoretical optimum. This error propagation factor can be calculated from the isotope system being measured and is dependent on the isotopic abundances in the natural sample and the isotopically enriched (spike) material. The error propagation factor is therefore the theoretical factor influencing the precision of the ratio measurements, as a function of the closeness to the calculated optimum spike. 

The specific activity SAi of the radioactive ( spike ) material before it is mixed with the sample is defined as... 

The recovery of mycotoxins from all extraction and cleanup procedures needs to be validated and this is usually done using carefully spiked material prepared from clean samples of the commodity under study. It must, however, be recognized that it is usually easier to recover compounds from spiked samples than it is to recover the same mycotoxins from naturally contaminated material, and for some mycotoxins there are available samples of naturally contaminated material the mycotoxin content of which has been established by international collaborative studies. The extraction and cleanup procedures will still usually provide a complex mixture requiring further separation before a specific physicochemical method for the quantitative or qualitative analysis of individual mycotoxins can be applied. This further separation is usually carried out by some form of chromatography. 

By using the terminology in eqn [2], where the subscript y denotes the enriched spike material and the subscript x denotes the natural isotopic abundance analyte in the sample, the isotope amount fractions can be expressed in the form of eqn [4]. If the spike isotope (B) is used as the reference isotope then the isotope amount fraction of isotope A to isotope B in the spike (Aspite) can be expressed as (eqn [5])... 

The isotopic abundance of the naturally occurring isotopes in the sample and the isotopically enriched analog in the spike must be well characterized. This can be a particular problem in inorganic ID-MS if isotopes of the analyte in the sample vary in nature, or are man-made (e.g., as a result of nuclear reactions). If this is the case the isotopic abundances must first be determined before IDA can proceed. Isotopically enriched spike compounds can normally be purchased with a certificate stating concentration and isotopic abundance however, it is good practice to at least verify the concentration. If the purity of the isotopically enriched analog has been characterized with sufficient accuracy and minimal uncertainty, the concentration can be calculated simply from knowledge of the masses of the compound and solvent employed. If the purity of the spike material is not certain, then reverse IDA is employed. In this case, the enriched spike material is treated as the sample and the isotopic abundance is modified by the addition of a certified standard of natural isotopic abundance that acts as the spike material. 

Although the technique is more expensive than radiometry, mass spectrometry is, however, the preferred method for IDA of samples of nuclear materials taken for accountability verifications. Traditionally, separated or highly enriched isotopes, absent from the samples or only present as minor isotopes, such as Pu, Pu or Th were used as spike material in isotope dilution mass spectrometry. IDMS is the standard operator method for the accountability of nuclear materials in input solutions of irradiated nuclear fuels. The method involves taking a representative sample from the input accountability tank and diluting it accurately 100-1,000-fold in a heavily shielded cell. A portion of the diluted solution is transferred to... 

Harrington et al. labeled a copper-containing protein rusticyanin (Rc) with Cu for use as a spike material in species-specific isotope dilution analysis, and... 

The same premise applied to safeguarding the material. "It is more valuable than gold," Price noted at a later meeting with the commissioners. "Banks know how to protect gold. We think these companies know how to protect this material." From a different perspective, Strauss challenged Price s argument. The value of the material did not bother Strauss, but possible diversion to a potential enemy did. "To the extent that weapons grade material or spiked material is involved, covert op-... 

With the exception of the certified standard ERM-AE 670, which can be directly used for spiking, isotopicaiiy labelled species must be characterised in terms of isotopic abundance and concentration before use. Isotopic abundance is usually determined using repeated GC-ICP-MS measurements of the isotopicaiiy labelled species solution after following the same derivatisation process as envisaged for the sample. Procedure blanks must be measured repeatedly in order to perform blank corrections, if necessary, on the spike material. Data evaluation is performed using integrated peak areas of all element isotopes. 

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