Analytical specifications

The analytical specifications must prescribe the ultimate performance of the total chromatographic system, in appropriate numerical values, to demonstrate the performance that has been achieved. 

The separation of the critical pair would require a minimum column efficiency and, so, the number of theoretical plated produced by the column must be reported. The 

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Unfortunately, some of the data that are required to calculate the specifications and operating conditions of the optimum column involve instrument specifications which are often not available from the instrument manufacturer. In particular, the total dispersion of the detector and its internal connecting tubes is rarely given. In a similar manner, a value for the dispersion that takes place in a sample valve is rarely provided by the manufactures. The valve, as discussed in a previous chapter, can make a significant contribution to the extra-column dispersion of the chromatographic system, which, as has also been shown, will determine the magnitude of the column radius. Sadly, it is often left to the analyst to experimentally determine these data. 

The analytical specifications must prescribe the ultimate performance of the total chromatographic system, in appropriate numerical values, to demonstrate the performance that has been achieved. The separation of the critical pair would require a minimum column efficiency and the number of theoretical plated produced by the column should be reported. The second most important requisite is that the analysis should be achieved in the minimum time and thus the analysis time should also be given. The analyst will also want to know the maximum volume of charge that can be placed on the column, the solvent consumption per analysis, the mass sensitivity and finally the total peak capacity of the chromatogram. The analytical specifications can be summarized as follows. 

1/ Column efficiency in theoretical plates. 2/ Analysis time. 

It is obvious that such a protocol would not be employed to design a column for a single analysis or even for a few dozen analyses. The optimization procedure entails a considerable amount of work and therefore, would only be justified for a routine analysis that was repetitive and would be carried 

LC COLUMN DESIGN-THE DESIGN PROCESS Packed Columns 

The design process involves the use of a number of specific equations (most of which having been previously derived and/or discussed) to identify the column parameters, the operating conditions and the resulting analytical specifications necessary to achieve a particular separation. The characteristics of the separation will be defined by the reduced chromatogram of the particular sample of interest. 

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Table 12. Chemical Compositions and Analytical Specifications for Chromate Corrosion Inhibiting Pigments...

The information contained in the three data bases provides the necessary information required to design the optimum column. In addition, once the column has been designed, and its properties defined, a complementary set of Analytical Specifications can also be calculated. Thus, the design protocol contains three data bases. Performance Criteria, Elective Variables and Instrument Constraints. 

These data bases will provide, first, the column specifications and, second, the analytical specifications. A diagram representing the overall design protocol is shown diagramatically in Figure 1. 

Unlike 2-thiazolidinethione and its simpler derivatives, the 3 - alkyl - 5 - hydroxy - 5 - (1,2,3,4 - tetrahydroxy -n- butyl) - 2 - thiazoli-dine thiones [which are specific, photometric reagents for Cu(ID] undergo rearrangement in the presence of Cu(II) ions, to form complexes of dithiocarbamate. The analytical specificity is explained by the inability of most other metal ions to effect this rearrangement (419). 

Fluorescence lifetime measurements can increase the analytical specificity when analyzing mixtures (1-4) and can indicate changes in chemical binding of the fluorophores under various environmental conditions (5). 

Residue study protocols typically either include quality specifications for analytical procedures or refer to a written analytical method that includes such specifications. The protocol for an LSMBS should also include analytical quality specifications, either directly or by reference to a method. Analytical specifications usually include minimum and maximum recovery of analyte from fortified control samples, minimum number of such fortifications per set of samples, minimum linearity in calibration, minimum stability of response to injection of calibration solutions, and limits of quantitation and of detection. 

The need for complete compositional analysis of additive packages in industrial plastics for both research and quality control applications has led to the development of numerous analyte-specific test procedures in recent years. 

Operator competence Analyte specific physico-chemical background... 

In coulometry, one measures the number of coulombs required to convert the analyte specifically and completely by means of direct or indirect electrolysis. 

The purity of the solid solute also has fundamental analytical implications. In general, the analytical procedure employed for determining the solubility should be specific for the solute of interest. For this purpose, an analyte-specific chromatographic method (such as high-performance liquid chromatography, HPLC) is preferred. Such a method will also enable the impurities and any possible decomposition products to be identified and quantified. 

A sensor array where different haptens are immobilized at well-defined areas on a plain glass surface has been developed [66], Using an automated flow injection system it was possible to incubate all areas on the chip with analytes, specific antibodies, secondary HRP-labeled antibodies, and CL substrate. Measurement of the light output via imaging performed with a CCD device allowed determination of the analytes present in the sample on the basis of the spatial localization of the CL signal. 

IVDs developed for use at a single site and offered commercially only at that site are considered laboratory testing services, in-house tests, or so-called "home brew" tests. These have historically been a widely used practice for test development, and a broad menu of tests is available in this mode. These tests are subject to CLIA regulation. If the test is performed by using commercially prepared and purchased active ingredients (so-called analytic specific reagents or ASRs), FDA does impose requirements on both the... 

Single-site IVD "Home brew" or "in-house" IVD made with an analyte specific reagent FDA requires labeling disclosing the "in-house" nature of the test but has no premarket review requirement CLIA requires analytical validation and quality control systems... 

Commercial reagents for use in "home brew" tests Analyte specific reagents FDA most 510(k) exempt blood bank and high-public health-risk reagents subject to premarket reviews CLIA oversight of laboratories using these reagents... 

FDA routinely requires analytical data on tests under review to demonstrate that they measure what they claim to measure. Analytical performance is usually directed at evaluating a test s accuracy or bias compared with a predicate or reference test, precision or repeatability, and analytical specificity and analytical sensitivity. 

An analytically sensitive test always detects analytes when they are present in specimens. An analytically specific test does not detect analytes when they are absent. The Task Force also recommended that laboratories providing a test for routine clinical use (after it had been developed) demonstrate their ability to provide analytically valid tests. These laboratories are regulated under the Clinical Laboratories Improvement Amendments of 1988 (Holtzman, 2000). 

Online SPE LC/MS/MS is commonly used for bioanalytical applications in the pharmaceutical industry. Column switching systems and TFC systems are easy to build and control. Sophisticated commercial systems and SPE cartridges are readily available. Compared to offline sample preparation, the online approach can save time and labor. However, the development of online SPE bioanalytical assays remains analyte-dependent. Generic methods can be applied to many analytes. For extremely hydrophobic, hydrophilic, and ionic analytes at normal pH range and analytes with a variety of hydrophobicity and pKa values, analyte-specific methods must be developed. An understanding of the chemistry of the analytes and SPE is critical. 

The alternative to FIA—MS performed in the standard addition mode is the quantification in FIA—MS—MS mode combined with a standard addition procedure. Here, the analytical specificity and the approach of a rapid analysis is combined, however, analysis time is expanded by a factor of 4.5 compared with FIA-MS. 

The RPIA technology has been enhanced in the Stratus CS system by utilization of a dendrimer-antibody complex in which the analyte-specific capture antibody is covalenty coupled onto a dendrimer. The test packs in the Stratus CS system include dendrimer-capture antibody complex reagent, the alkaline phosphatase labeled antibody conjugate reagent, the substrate-wash reagent and a piece of glass fiber filter paper as the solid phase. Preparation and unique properties associated with these dendrimer-coupled antibody complexes are described below. 

Table 19.2 Sensitivity determination with analyte-specific primary antibody in the form of a dendrimer complex or a double-antibody immune complex...

The dominant attribute that has driven interest in fluorescent conjugated polymers (CPs) sensory materials is their ability to produce signal gain in response to interactions with analytes. The increased sensitivity (amplification) is derived from the ability of a conjugated polymer to serve as a highly efficient transport medium of electronic excitation. Analyte specificity in CP-based sensors results from the covalent or physical integration of receptors, imprinting, and/or the CP s overall electrostatic and chemical characteristics. The observed amplification is a... 

Difficulties are encountered in the qualitative and quantitative analysis of carbohydrate mixtures because of the structural and chemical similarity of many of these compounds, particularly with respect to the stereoisomers of a particular carbohydrate. As a consequence, many chemical methods of analysis are unable to differentiate between different carbohydrates. Analytical specificity may be improved by the preliminary separation of the components of the mixture using a chromatographic technique prior to quantitation and techniques such as gas-liquid and liquid chromatography are particularly useful. However, the availability of purified preparations of many enzymes primarily involved in carbohydrate metabolism has resulted in the development of many relatively simple methods of analysis which have the required specificity and high sensitivity and use less toxic reagents. 

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