Selectivity and specificity in analytical chemistry. General considerations and attempt of a definition and quantification

Selectivity and specificity are performance characteristics of analytical methods which are frequently used in analytical literature. In general, the terms are applied verbally and a quantification of selectivity and specificity is given rarely. Excepted are methods like chromatography and ISE sensoring which use individual quantities such as selectivity coefficients, indices and other parameters to characterize analytical procedures and systems. Here a proposal is given to characterize selectivity and specificity quantitatively by relative values in a range of 0 to 1 expressing so a certain degree of selectivity and specificity. By examples it will be shown that the derived quantities characterize analytical methods and problems in a plausible way.     

Selectivity and specificity in analytical chemistry. General considerations and attempt of a definition and quantification

Selectivity and specificity are performance characteristics of analytical methods which are frequently used in analytical literature. In general, the terms are applied verbally and a quantification of selectivity and specificity is given rarely. Excepted are methods like chromatography and ISE sensoring which use individual quantities such as selectivity coefficients, indices and other parameters to characterize analytical procedures and systems. Here a proposal is given to characterize selectivity and specificity quantitatively by relative values in a range of 0 to 1 expressing so a certain degree of selectivity and specificity. By examples it will be shown that the derived quantities characterize analytical methods and problems in a plausible way.     

Adaptive multicomponent analysis by genetic algorithms

The applicability of genetic algorithms for solving multicomponent analyses is systematically examined. As a genetic algorithm (GA), the basic proposal of Goldberg is implemented in a straightforward manner to simulate multicomponent analyses in analogy to the well-established UV-vis or IR methods, especially multicomponent regression. The main focus of the study is to investigate the behavior of the genetic algorithm in order to compare it with the well-known behavior of multicomponent regression. A remarkable difference between the two methods is that the genetic algorithm method does not need any calibration procedure because of its pure searching characteristic. As important features of multicomponent systems, the degree of signal overlap (selectivity), the behavior of systems with known and unknown component numbers and qualities, and linear as well as nonlinear relationships between the analytical signal and concentration are varied within the simulations. According to multicomponent regression, recovering concentrations by a genetic algorithm is of limited applicability with the exception of systems at a low degree of signal overlap. On the other hand, the recovery of a probe spectrum in the analytical process always gives satisfactory results independent of the features of the probe system. The genetic algorithm obviously shows autoadaptive behavior in probe spectrum recovery. The quality and quantity of the resulting components may dramatically differ from the given probe, although the resulting spectrum is nearly the same. In such cases, the resulting component mixture can be interpreted as an imitation of the probe. As well probe spectra, theoretically designed spectra can also be autoadapted by genetic algorithms. The only limitation is that the desired spectrum must, of course, be incorporated into the search space defined by the involved components. Furthermore, a spectral signal is only one single property of a chemical compound or mixture. Because of the nonlinear search characteristic of genetic algorithms, any other chemical or physical property can also be treated as a desired property. Therefore, the conclusion of the study is well-founded that an old challenge of applied chemistry, namely, the development of new chemical products with desired properties, seems to be reachable under the control of genetic algorithms.     

Two novel procedures for automatic resolution of two-way data from coupled chromatography

In order to deal with the problem of simultaneous qualitative and quantitative analysis of the complicated samples in analytical chemistry, two novel procedures for automatic resolution of two-way data from coupled chromatography were developed in this work. The first can be used to determine automatically the number of components involved in a certain peak cluster. The second method is an iterative resolution procedure named the stepwise key spectrum selection which has also been developed to resolve automatically the spectra and chromatographic profiles of every component in the peak cluster investigated. The results obtained from simulated and real data show that the proposed methods perform fairly well.     

The use of ATR-IR to identify the components separated by paper chromatography

Summary A modified procedure for the identification of separated components in paper chromatography using attenuated total reflection infrared spectroscopy is described. This combined method, though inferior in sensitivity and resolution to the modern separation systems interfaced with sophisticated analytical instruments, is simple, relatively less expensive and suitable for routine analysis of components like polymer additives.     

Variable flow liquid chromatography-tandem mass spectrometry and the comprehensive analysis of complex protein digest mixtures

A microscale, variable flow, liquid chromatography-electrospray ionization source has been coupled to an ion trap mass spectrometer and used to analyze both simple and complex protein mixtures. By using an analytical technique described as “peak parking,” the sample analysis times for components eluting from a liquid Chromatograph can be greatly increased without sacrificing Chromatographic resolution. This was achieved by instantaneously reducing the column head pressure (and hence, flowrate) under peaks of interest to prolong their elution from the tip. With the technique, it is possible to perform manual parent ion selection, higher resolution narrow mass range scans, and multiple stages of tandem mass spectrometry (MS² and MS³) within the context of a single eluting peak. Benefits normally associated with an off-line tandem mass spectrometry analysis, such as the optimization of collision induced dissociation parameters and the analysis of more than one charge state, can now be obtained with the increased sensitivity and selectivity afforded by the chromatography. The utility of this method for the comprehensive analysis of complex mixtures was illustrated with the analysis of an in-gel protein digest mixture derived from a single spot from a two-dimensional electrophoresis gel. In addition to the expected enzyme digestion products, other peptides derived from nonspecific cleavage by the enzyme and protein impurities, as well as those having oxidized, derivatized, or deamidated amino acid residues were fully characterized.     

Semi-automated peak trapping recycle chromatography instrument for peak purity investigations

A peak trapping recycle chromatography system has been developed and optimized for peak purity assessment of active pharmaceutical ingredients analyzed by high performance liquid chromatography (HPLC). After being analyzed using a reversed phase analytical column, peaks of interest are trapped and are subsequently introduced to a recycle chromatography system. In addition to the increased effective length afforded the recycling system, the small selectivity difference between the analytical and recycling methods help separate potential impurities under the main peak. For more difficult to separate components, the increased efficiency of recycle chromatography provides the necessary resolution. Over 227,000 theoretical plates have been obtained in the recycle dimension for some compounds. The sensitivity of the system fell short of the target (0.1%), but it did show sensitivity (0.5%) comparable to other peak purity techniques commonly used in the pharmaceutical industry. The recovery and repeatability have also been shown to be adequate for peak purity assessment. The system has also been automated using a Visual Basic macro, simplifying the interface allowing it to be used as an open access instrument.     

Estimation of limits of detection and determination in X-ray fluorescence analysis by the dependence of the relative standard deviation on analyte concentration

An experimental dependence of the relative standard deviation on analyte concentration of hyperbolic type, characterizing the precision of quantitative chemical analysis, was used to estimate the limits of detection and determination in the X-ray fluorescence analysis. A method is proposed for the determination of their values using the approximation of the experimental dependence of the relative standard deviation on the analyte concentration by a power function. The choice of the values of the relative standard deviation, being criteria for the estimation of these limits, is substantiated. A concept of the limits of detection and determination of an analytical procedure is formulated, according to which the limit of detection of an analytical procedure is an objective value depending only on the precision of determinations, and the limit of determination of an analytical procedure is a subjective value depending not only on the precision of determinations but also on the requirements to their limiting (admissible) accuracy. The limits of detection and determinations of an analytical procedure found by this approach completely characterize the possibilities of an analytical procedure in determining low concentrations of analytes. The proposed approach can be used for the estimation of the limits of detection and determination of analytical procedures and in other methods of chemical analysis with the hyperbolic dependence of the relative standard deviation on the analyte concentration.     

Liquid exclusion adsorption chromatography, a new technique for isocratic separation of nonionic surfactants. III. Two-dimensional separation of fatty alcohol ethoxylates

In this paper, a novel procedure for qualitative and quantitative analysis of the two-dimensional data obtained from GC-MS is investigated to determine chemical components of essential oils in Cortex Cinnamomi from four different producing areas. A new method named iterative optimization procedure (IOP) specially used to resolve embedded peaks is also developed. With the help of IOP and other chemometric techniques, such as heuristic evolving latent projections, evolving factor analysis, sub-window factor analysis and orthogonal projection resolution, and etc., the detection of the purity of chromatographic peaks can be first addressed, and then the overlapping peaks are resolved into the pure chromatogram and mass spectrum of each component. The similarity searches in the MS database are finally conducted to qualitatively determine the chemical components. The results obtained showed that the accuracy of qualitative and quantitative analysis could be greatly enhanced by chemometric resolution methods. The chemometric resolution techniques upon the two-dimensional data can be quite promising tools for the analysis of the complex samples like traditional Chinese medicine.     

Some remarks on the information power and selectivity of analytical processes

The consistency of the equation given by Shannon for the transfer of maximum amount of information in one channel, and the equation deduced by Kaiser for the expression of the information power of analytical methods is shown. The fundamental difference between the maximum amount of information obtainable by an instrument and that by an analytical procedure is reported. In the second case the selectivity of the analytical procedure also has to be taken into consideration.     

The modular analytical procedure and validation approach and the units of measurement for genetically modified materials in foods and feeds

Food and feed analysts are confronted with a number of common problems, irrespective of the analytical target. The analytical procedure can be described as a series of successive steps: sampling, sample processing, analyte extraction, and ending, finally, in interpretation of an analytical result produced with, e.g., real-time polymerase chain reaction. The final analytical result is dependent on proper method selection and execution and is only valid if valid methods (modules) are used throughout the analytical procedure. The final step is easy to validate-the measurement uncertainty added from this step is relatively limited and can be estimated with a high degree of precision. In contrast, the front-end sampling and processing steps have not evolved much, and the corresponding methods are rarely or never experimentally validated according to internationally harmonized protocols. In this paper, we outlined a strategy for modular validation of the entire analytical procedure, using an upstream validation approach, illustrated with methods for genetically modified materials that may partially apply also to other areas of food and feed analyses. We have also discussed some implications and consequences of this approach in relation to reference materials, measurement units, and thresholds for labelling and enforcement, and for application of the validated methods (modules) in routine food and feed analysis.     

Isotachophoretic analysis of pharmaceutic formulations with different matrices: Determination of nicotinic acid in capsules,tablets, dragees,ointments and tinctures

The selectivity of isotachophoresis is investigated concerning the quantitative determination of an anionic agent in a variety of pharmaceutic application forms with different matrices. As a result, no interferences caused by matrix components were observed in any case. Therefore sample pretreatment can be neglected. The simultaneous quantitation of other anionic constituents, present in the formulations, is possible. The method is evaluated by other analytical characteristics besides selectivity, like precision, accuracy and time of analysis. The contributions of aliquotation and isotachophoretic measurement to the total error were determined by analysis of variance. Systematic errors, due to adsorption of the analyte on solid matrix material were examined by standard addition. The results of the analytical characteristics indicate the excellent applicability of isotachophoresis for the routine analysis of anionic analytes in pharmaceutic preparations     

Chiral analysis by mass spectrometry using the kinetic method in flow systems

Chiral analysis is an important task of analytical chemistry. Besides separation techniques, mass spectrometry can be applied in this field. One mass spectrometric approach is based on Cooks' kinetic method. The method was successfully applied in a static system in which the concentration of the analyte as well as the chiral selector solution was constant during the experiment. The application of the kinetic method in dynamic systems (changing concentration of analyte) is presented. Such systems allow the speeding up of the analytical process (flow injection analysis (FIA)) or the use of the kinetic method for chiral detection after liquid chromatographic separation.The influence of the concentration of the components of the chiral selector solution as well as its flow rate on the recognition of enantiomers was evaluated. A new procedure for correction for the differences between ratio of enantiomers in the liquid phase and their observed ratio in the gas phase is also described. A significant improvement in accuracy using this procedure was achieved. Applicability of the method was demonstrated in the analysis of amino acids using FIA as well as HPLC/MS. After an achiral separation of leucine and isoleucine, chiral mass spectrometric detection was successfully used for enantiomeric recognition.     

Use of optimization software to determine rugged analysis conditions in high-performance liquid chromatography

The ruggedness evaluation of an analytical method is now generally required for further validation. By considering ruggedness at an early stage of method development, major disappointments and amount of work could be avoided. This work shows that the optimization software OSIRIS can be helpful for the chromatographer during a method development, as it takes into account the method ruggedness. The ruggedness of the analysis conditions is then evaluated all along the selectivity optimization procedure. This optimization software belongs to the interpretive methods that consist of predicting the optimum conditions by modeling first the solute retention over the parameter space using a minimum number of preliminary runs. The choice of a response function is studied. This response function must be able to take into account several individual criteria: analysis time, minimal resolution and ruggedness of each parameter. Some optimum separations, determined using a ruggedness criteria or not, are given and compared in terms of long term repeatability.     

Flow-injection determination of zidovudine in plasma samples using multivariate curve resolution

This paper describes a spectrophotometric flow-injection method for the determination of an antiretroviral drug (zidovudine) in plasma samples. The main goal of this study is the development of feasible analytical method to monitor the plasmatic drug levels in a rapid and simple way as an alternative to chromatographic procedures. The flow-injection system proposed consists of a two-channel manifold with the injection of the sample into an acid carrier and on-line generation of a pH-gradient. The corresponding data are monitored over time using a diode array spectrophotometer. The discrimination of zidovudine species from plasma components is accomplished through chemometric data analysis based on the zidovudine features on both spectral and time domains. A pretreatment procedure consisting of liquid-liquid extraction is used for sample clean-up. However, despite the pretreatment, noticeable amounts of unknown substances acting as interferences are still present in the extracts. As relevant analytical parameters, the analyte recovery in the extraction process is 101% at 5.3 μg mL⁻¹ and the detection limit is 0.013 μg mL⁻¹. Multivariate curve resolution with alternating least squares is used to recover the analyte profiles for its further determination. As a result, the quantification of zidovudine in plasma can be accomplished even in the presence of plasma components with overall prediction error below 3%.     

Investigations of the chemical homogeneity of solids

Chemical homogeneity is a relative property of solid-state materials; it depends not only on their ultimate purposes, but also on the parameters of the analytical procedure applied in the investigation. These features are often given inadequate attention in the characterization of solid materials. A statistical model for representative sampling is used here as the starting point to derive relationships which demonstrate quantitatively the increasing rigour of the homogeneity test with increasing spatial resolution and precision of the analytical procedure. It is also shown that chemical homogeneity can be confirmed at a calculated risk, when the tests do not show the material to be significantly inhomogeneous and when maximum permissible deviations in concentration can be given for a practical application.     

"Orthogonal" separations for reversed-phase liquid chromatography

A general procedure is proposed for the rapid development of a reversed-phase liquid chromatographic (RP-LC) separation that is "orthogonal" to a pre-existing ("primary") method for the RP-LC separation of a given sample. The procedure involves a change of the mobile-phase organic solvent (B-solvent), the replacement of the primary column by one of very different selectivity, and (only if necessary) a change in mobile phase pH or the use of a third column. Following the selection of the "orthogonal" B-solvent, column and mobile phase pH, further optimization of peak spacing and resolution can be achieved by varying separation temperature and either isocratic %B or gradient time. The relative "orthogonality" of the primary and "orthogonal" RP-LC methods is then evaluated from plots of retention for one method versus the other. The present procedure was used to develop "orthogonal" methods for nine routine RP-LC methods from six pharmaceutical analysis laboratories. The relative success of this approach can be judged from the results reported here.     

Criteria for judging the quality of separation in chromatograms containing solvent peaks

Summary Formal procedures used for optimizing chromatographic selectivity require objective, numerical functions to assess the quality of each chromatogram obtained during the process. Many of such optimization criteria have been suggested for chromatograms in which all or a number of well-shaped peaks need to be separated. This paper describes a method by which resolution can be measured in chromatograms in which very large, highly non-symmetrical background peaks,e.g. typical solvent peaks, are present. Using this method, various optimization criteria can be calculated. The method is evaluated using a simulation program which constructs chromatograms from the recorded profiles of individual experimental peaks. It is also demonstrated for use in an experimental optimization procedure in reversed-phase liquid chromatography.     

Polycyclic aromatic hydrocarbons in waste waters and sewage sludge: Extraction and clean-up for HPLC analysis with fluorescence detection

Summary A modified solid-phase extraction technique using sonication of the adsorbent material instead of the elution normally applied has been compared with two conventional liquid-liquid extraction procedures for the determination of the 16 EPA PAHs in municipal waste waters by means of HPLC coupled with fluorescence detection. Liquid-liquid extraction with cyclohexane proved to be the most efficient and simplest procedure. Clean-up of the waste-water extracts was not considered necessary, because of the high chromatographic resolution of the column and the selectivity of the fluorescence detector. Different organic solvents were also compared for ultrasonic extraction of PAHs from sewage sludge. The best results were obtained by use of acetonitrile. Clean-up of sewage-sludge extracts was not found necessary for accurate quantification of the major PAH components with fluorescence detection. The precision of the whole analytical procedure from extraction to the final determination of PAHs was satisfactory for both waste-water and sewage-sludge samples.     

Purification and analysis of partially alkylated cyclodextrins by liquid and gas chromatography

Complex mixtures of partially alkylated cyclodextrins can be analyzed by both HPLC and high temperature capillary GC. Because of the limited efficiency of LC, suitable analytical and preparative separations can be achieved only with systems of carefully optimized selectivity. Using LC it has been possible to isolate and purify single cyclodextrin species from very complex mixtures of components which contain unreacted hydroxyl groups in addition to the alkoxy groups. Analysis of the reaction mixtures and of fractions taken from LC separations can be performed with advantage by high resolution capillary GC at high temperatures between 300 and 400 °C. The thermal stability of partially alkylated cyclodextrins in high temperature GC is considerably increased by trimethylsilylation of the free hydroxyl groups. Fast atom bombardment mass spectrometry and proton NMR were used to identify species isolated from the preparative LC separations.