Internal Reference Standards techniques

Indeed, there are potentially several different levels to approach standardization of IHC For the greatest scientific rigor, it is required to perform serial experiments based upon the hypothesis mentioned above to investigate and validate, if possible, standardization of IHC based on the AR technique (Chapter 5 in detail). Another approach, that may be combined with AR, is the systematic development of Quantifiable Internal Reference Standards (QIRS) that will allow assessment of the degree of protein degradation... 

The sample was prepared by the neat technique on KBr plate or by the KBr pellet technique. The proton nuclear magnetic resonance (NMR) spectra were obtained at 60 MHz with a JEOL-PMX 60 spectrometer. Deuterochloro-form and carbon tetrachloride were used as solvents with tetramethyl-silane (TMS) as the internal reference standard. The X-ray diffraction patterns of the powdered samples were taken in the region of 5 to 3 o by a Rigakudenki Model DC-8 X-ray diffractometer, using Ni-filtered Cu-Ka radiation. 

If the precision offered by the external standard technique is not adequate, it is necessary to use an internal standard. In this procedure, a known amount of a reference substance, not originally present, is introduced into the sample. This will result in the appearance of an additional peak in the chromatogram of the modified sample and any variations in the injections volume will equally affect the standard and the test compounds. 

A single measurement of a calibration sample can give the concentration of the test solution by a simple ratio. This is often done in techniques where a calibration internal standard can be measured simultaneously (within one spectrum or chromatogram) with the analyte and the system is sufficiently well behaved for the proportionality to be maintained. Examples are in quantitative nuclear magnetic resonance with an internal proton standard added to the test solution, or in isotope dilution mass spectrometry where an isotope standard gives the reference signal. For instrument responses As and /sample for internal standard and sample, respectively, and if the concentration of the internal standard is Cjs, then... 

For the internal standard method, a substance is added at the earliest possible point in the analytical scheme. This compensates for sample losses during extraction, cleanup, and final chromatographic analysis. There are two variations in the use of the internal standard technique. One involves the determination of response factors which are the ratios of the analyte peak response to the internal standard peak response. The second is referred to as response ratios which are calculated by dividing the weight of the analyte by the corresponding peak response. 

An in vitro bioassay can be designed in several ways, but requires statistical validity. A one point assay is not valid. The bioassay should be designed to consider factors that introduce variability, and the analysis should test such variability. A measurement series of a test sample should be compared to an equivalent series of the reference material, carefully considering the comparisons between the linear portions of the dose-response curves (Mire-Sluis et al., 1996). To test validity of a bioassay inter- and intra-assay variability should be considered in both preparation, and in the case of multiwell plates, the variability between each plate. To reduce the positional effect in plate tests, it is advisable to distribute the points on the curves randomly and also to include a reference standard in each plate (Gaines-Das and Meager, 1995). One of the most widely used techniques to validate a bioassay s performance is to include internal duplicates. The data arising from the comparison can be important in assessing the test s variability. 

The direct determination of trace elements (Al, Ba, Cu, I, Mn, Mo, Pb, Rb, Se, Sr, and Zn) by ICP-MS in powdered milk was reported [14]. Samples were diluted with a 5 or 10 percent (v/v) water-soluble, mixed tertiary amine reagent at pH 8. This reagent mixture dissociated casein micelles and stabilized liquid phase cations. Mass intensity losses were not observed. The quantitative ICP-MS procedure was applied the standard additions method with a Y internal reference. This direct technique is as fast as the slurry approach without particle size effects or sensitivity losses. 

Yet, another feature that becomes available with the fast sequential mode of operation in HR-CS AAS is the use of the reference element technique, that is, the use of an internal standard. This technique has rarely been described in LS AAS [3] for a number of obvious reasons. Firstly, the technique requires a dual-or multi-channel spectrometer, and there have been only very few spectrometers of this type available commercially over the past decades. Secondly, the reference element technique is ideally suited to correct for nonspecific interferences, such as transport interferences, but it is notoriously difficult to find an appropriate reference element for element-specific interferences. Thirdly, the most successful multi-channel LS AAS equipment, the Perkin-Elmer Model SIMAA 6000, which has been available for a number of years, was designed for ET AAS only, a technique that does not typically exhibit nonspecific interferences. The number of publications using this technique is therefore very limited. 

In the physical area the validity of measurements is established by means of an unbroken chain of comparisons from the working standards in day-to-day use through reference standards to the national and international primary standards, using recognised measurement techniques and methods. In this way the measurements are traceable back to the primary standards. 

The method is based on the addition of a standard reference (internal standard) that is detected at a different wavelength from the analyte. The reference standard is added at the same concentration to samples and standards and diluted to mark in a volumetric flask. This technique uses the signal from the internal standard to correct for matrix interferences and is used with respect to precision and accuracy as well as eliminating the viscosity and matrix effects of the sample. 

In vitro assays do not use any whole-cell or animal-based components. The fibrin clot lysis assay, as established for tissue plasminogen activators and described for alteplase in the USP, is an example of this type of potency testing [5]. By means of defined standard materials, a fibrin clot is formed and the time to complete lysis is characterized as measure of potency, compared to a reference standard with defined activity. The LAL-test is a well-established and internationally harmonized in vitro alternative to detect or quantify bacterial endotoxins, using Limulus amebocyte lysate (LAL) obtained from the aqueous extracts of circulating amebocytes of horseshoe crab (Limulus polyphemus or Tachypleus tri-dentatus) which has been prepared and characterized appropriately [5]. Two types of technique may be used for this test gel-clot techniques, which are based on gel formation and photometric techniques. 

However, even with this technique we must take into account the possibility of medium effects on chemical shifts. So far two techniques have been used to compensate for this effect one procedure uses the intramolecular shift between two groups (e.g. CH2 and CH3 in an ethyl derivative), and the other the intermolecular shift relative to a suitable chosen reference (in most cases the trimethylammonium ion) as an internal reference. The first method has the advantage that it does not require the introduction of any other substances into the solutions, but it suffers from the disadvantage that the relative intramolecular shifts are small. The use of an internal standard, whose chemical shift is also affected by the solvent, may alter the acidity of the solution, although the use of small amounts (0-05 m) of trimethylammonium sulfate should give negligible changes. 

The echo-peak technique, which simulates the use of an internal standard, is based on two consecutive injections of a reference standard and a sample extract within the same LC analysis. The first and second injections are made to either pass through or bypass a pre-column, respectively. This results in a short difference in retention times between a known amount of reference standard, which is also the analyte of interest, and an analyte from a sample. As a result, the reference elutes close to the analyte. The peak from a reference is referred to as an echo-peak, and the peak from a sample is called a sample peak (Fig. 6.6). Provided that the retention times of these two peaks are close enough, both the reference and the analyte encounter... 

Quality control (QC) refers to the operational techniques and activities that are used to fulfill requirements for quality. Internal quality control comprises the routine practical procedures that enable the analytical chemist to make a decision on whether to accept a result or a group of results as fit for purpose, or reject them and repeat the analysis. Tools for quality control include the use of reference standards and certified reference materials, the use of positive (spiked or incurred) and negative control samples and control charts, replicate analyses, and proficiency tests. Quality control in the laboratory is discussed in more detail in Section 10.5 of this chapter. 

All the methods of analysis described above, with the exception of some of the mass spectrometry applications, measure concentrations relative to a standard of known composition or to a calibration curve, drawn on the basis of standards of known composition. The standards used in the construction of calibration curves are either ultra-pure chemical reagents or, where matrix effects are important in some rock samples, well-analysed in-house samples and international reference samples (Govindaraju, 1984 Abbey, 1989). In either case the standards should be analysed using the most precise technique possible. Qearly the accuracy of the final analysis depends upon the accuracy of the standards used in calibration and systematic effbis can easily be introduced. 

The colloidal state inevitably brings about difficulties for the experimentalist when separation of the disperse phase from the dispersion medium is needed. This is the case when the speciation and concentration of only the free soluble species have to be determined. Separation of the ionic solution from the small colloidal particles for conventional chemical analysis is nontrivial, although separation techniques such as ultracentrifugation, dialysis, and field-flow fractionation have been successfully used. If the soluble species of interest have an active nuclear spin, the liquid NMR technique wiU constitute an alternative and simpler way to characterize and quantify those species without being affected by the disperse phase. An exception is the case where the colloidal species gives a signal that fully overlaps the sharp resonance of the solution entity. As NMR is quantitative, the absolute concentration of the species can be estimated based on an internal reference of known concentration but different chemical shift relative to the sample signals. Alternatively, a calibration curve can be established from a set of external standard solutions (preferably the same substance found in the sample) measured under the same experimental NMR conditions as those applied to the sample. 

Quality control is of the utmost importance in the case of mineral analyses because of the low concentrations of the elements normally found in foods and the ubiquitous presence of significant levels of many of them in the environment. In addition to the standard techniques of working in a clean laboratory to reduce the potential for accidental contamination to a minimum, it is essential that procedures be validated and results checked against appropriate certified reference materials (CRMs). CRMs for most of the trace and other minerals of interest in foods are available from international reference centers such as the Community Bureau of Reference of the European Union, the National Institute of Standards and Technology of the United States, and the International Atomic Energy Agency in Vienna. 

This section provides a simplified introduction to the methods by which calibration solutions containing analytical (or reference) standards, with or without internal standards, are used in practice to measure amounts of analytes in unknown samples. The main deficiency of this brief account is its lack of any attempt to take into account experimental uncertainties (both random and systematic) and thus the level of confidence in the results thus obtained. While this book is directed towards analyses in which mass spectrometry is used as the chromatographic detection technique, most of the following discussion is applicable also to other detectors the main exception concerns use of isotope-labeled surrogate internal standards, for which only mass spectrometry can provide adequate detection. A much more complete account of this material, including a discussion of the associated random and systematic errors, is given in Section 8.5. 

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