Analytical Specificity and Interference

In relation to immunoassays, interference from proteins (usually antibodies) should be recognized. Ismail et aft found in a survey comprising more than 5000 TSH results that interference occurred in 0.5% of the samples, leading to incorrect results that in a majority of the cases could have changed the treatment. Rotmensch and Cole described 12 patients in whom a diagnosis of postgestational choriocarcinoma was made based on false-positive test results for 

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When the solution sample is suitably diluted, usually in simple media, the analyte may be quantitated in seconds. Automation is widely and relatively inexpensively available. Atomic spectroscopy is inherently very specific and interferences that will reduce the bias below the level of the precision are few. Those interferences that exist are well characterized and are generally controlled in a routine manner. The net result of all this is that flame AAS should be used in every situation where it is applicable. 

Nonlinear techniques have been used to overeome some of the drawbacks of conventional Raman spectroscopy, particularly its low dficienev, its limitation to the visible and near-ultraviolet regions, and its susceptibility to interference from fluorescence. A major disadvantage of nonlinear methods is that they lend to be analyte specific and often require several different tunable lasers to be applicable lo diverse species. I o dale, none of the nonlinear methods has found widespread application among nonspccialisls. However, many of these methods have shown considerable promise. As less expensive and more routinely useful lasers become available, nonlinear Raman methods, particu-larlv CARS, should become more widely used. 

There are various methods of mineral determination available, and they can be used according to the analytical characteristic that best suits the objectives of the analyst accuracy, sensitivity, detection limit, specificity, and interferences. Other factors to take into account are the costs of the complete analysis, instrumental availability, and time necessary for analysis. 

An analytical method vahdation study should include demonstration of the accuracy, precision, specificity, limits of detection and quantitation, linearity, range, and interferences. Additionally, peak resolution, peak tailing, and analyte recovery are important, especially in the case of chromatographic methods (37,38). 

Specific extraction methods are used to prepare the analyte for immunoassay by freeing the analyte fromboth specific and nonspecific interferences. Supercritical fluid extraction has been used to decrease the amount of solvent waste generated. Solid-phase extraction has gained popularity, and many different supports are available. One promising extraction and concentration method is immunoaffinity chromatography, which will be addressed later. 

Third, the bulk of the items in Table 1 address method performance. These requirements must be satisfied on a substrate-by-substrate basis to address substrate-specific interferences. As discussed above, interferences are best dealt with by application of conventional sample preparation techniques use of blank substrate to account for background interferences is not permitted. The analyst must establish a limit of detection (LOD), the lowest standard concentration that yields a signal that can be differentiated from background, and an LOQ (the reader is referred to Brady for a discussion of different techniques used to determine the LOD for immunoassays). For example, analysis of a variety of corn fractions requires the generation of LOD and LOQ data for each fraction. Procedural recoveries must accompany each analytical set and be based on fresh fortification of substrate prior to extraction. Recovery samples serve to confirm that the extraction and cleanup procedures were conducted correctly for all samples in each set of analyses. Carrying control substrate through the analytical procedure is good practice if practicable. 

The principal advantages of RIA are its sensitivity, specificity and simplicity of operation. There are three main disadvantages. Firstly, the lengthy development periods for new methods often result from the difficulties of producing the specific binding agent. Secondly, cross-reactions with other molecules similar to the analyte can sometimes interfere. Thirdly, poor precision can result unless careful control of experimental conditions is employed to ensure reproducible binding reactions. Relative precisions of 1-3% are typical. 

Optical fiber detectors (OFD) are devices that measure electromagnetic radiation transmitted through optical fibers to produce a quantitative signal in response to the chemical or biochemical recognition of a specific analyte. Ideally, an OFD should produce a specific and accurate measurement, continuously and reversibly, of the presence of a particular molecular species in a given sample medium. Additionally, OFD should pro vide maximum sensitivity and minimal interferences fromsuperfluous ions or molecules to obtain low detection limits. Other attractive features include the miniaturization of the fiber s tip to accommodate single-cell analysis and portable instrumentation to allow in situ analysis. 

Many dyes complex with their primary analyte due to attractive forces such as ionic charges. These charges are susceptible to nonspecific complexation with interfering analytes with characteristics similar to those of the primary analyte. Both the primary and interfering analytes compete for complexation with the same site. The NIR dyes may be synthesized with specific functional groups that will bond more specifically to an analyte. For instance, an isothiocyanate group forms very stable thiourea chemical bonds with proteins or an amino-modified DNA oligomer. The introduction of more specific and reversible functionalities on the dye molecule should minimize the interference of extraneous molecules or ions. 

One of the most important advantages of HPLC over spectrophoto-metric methods lies in its specificity and selectivity due to its separation capability. Through chromatographic separations, the analytes of interest can be detected and quantified without interference from the typical matrix that includes excipients, antioxidants, preservatives, and dissolution media. Ion-pair HPLC was used to monitor the dissolution of pentamidine from EVA sustained-release film where polymeric matrices could create significant bias if a spectrophotometric method were used. Due to their strong UV absorbance, the antioxidants and preservatives (e.g., BHA, BHT, ascorbic acid and propyl gallate) are often the major... 

It is beneficial to develop generic methods, this means methods that are already validated for some aspects, e.g., EOF stability and freedom of matrix interferences. Generic methods that are suitable to separate a number of analytes can be found in CE, especially due to the high separation efficiency. Some aspects, like specificity and repeatability, are still analyte specific, but the entire method validation is substantially speeded up when a generic method already exists. 

The analytic principles that have been applied to accumulate air quality data are colorimetry, amperometry, chemiluminescence, and ultraviolet absorption. Calorimetric and amperometric continuous analyzers that use wet chemical techniques (reagent solutions) have been in use as ambient-air monitors for many years. Chemiluminescent analyzers, which measure the amount of chemiluminescence produced when ozone reacts with a gas or solid, were developed to provide a specific and sensitive analysis for ozone and have also been field-tested. Ultraviolet-absorption analyzers are based on a physical detection principle, the absorption of ultraviolet radiation by a substance. They do not use chemical reagents, gases, or solids in their operation and have only recently been field-tested. Ultraviolet-absorption analyzers are ideal as transfer standards, but, as discussed earlier, they have limitations as air monitors, because aerosols, mercury vapor, and some hydrocarbons could, interfere with the accuracy of ozone measurements made in polluted air. 

Selectivity is often referred to as the specificity of an analytical method and is a measure of the discriminating ability of the technique. The general requirement for specificity is that the method should be capable of unambiguously determining the compounds of interest in the presence of impurities, degradation products and other sample matrix components. A specificity study often involves accelerated degradation studies to ensure all degradation products will not interfere and the collection of likely process impurities. Often a placebo sample is assayed to check for interference fi om the sample matrix. 

Sensors or analyzers exist for some of the priority analytes, such as 09, pH, and N03 . The challenge in these cases is to improve sensor stability, response rates, or lifetime. However, for most of the priority analytes, there is no existing sensor or analyzer system that will operate for long time periods without operator intervention. The development of sensors for most of these analytes, such as chlorofluorocarbons or dissolved iron, must circumvent the difficulties posed by low analyte concentrations or interference from other dissolved material. Development of specific sensing chemistry is the ultimate means of circumventing these problems. 

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