Analyte quantification

Dolan JW, Snyder LR. Gradient Elution Chromatography. In Meyers, RA (ed). Encyclopedia of analytical chemistry. Chichester John Wiley Sons, 2000 11342-60. 

Eiceman GA, Gardea-Torresdey J, Overton E, Carney K, Dorman F. Gas Chromatography. Anal Chem 2002 74 22771-80. 

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Famoxadone, IN-JS940, and IN-KZ007 residues are measured in soil (p-g kg ), sediment (p-gkg ), and water (pgL ). Quantification is based on analyte response in calibration standards and sample extract analyses determined as pg mL Calibration standard runs are analyzed before and after every 1 samples in each analytical set. Analyte quantification is based on (1) linear regression analysis of (y-axis) analyte concentration (lagmL Q and (x-axis) analyte peak area response or (2) the average response factor determined from the appropriate calibration standards. The SLOPE and INTERCEPT functions of Microsoft Excel are used to determine slope and intercept. The AVERAGE and STDEV functions of Microsoft Excel are used to determine average response factors and standard deviations. 

Achiral-chiral LC-LC is most often used to separate the desired analyte from interfering components, such as matrix components, metabolites, excess derivatiza-tion reagent, or other impurities. Separating such interferents from the analyte allows for better analyte quantification or enantiomeric ratio determination. Also, achiral columns are seen as a way to protect the more expensive chiral columns from matrix components that might become irreversibly retained and deteriorate column performance. Short achiral columns (trap columns) are sometimes used to reconcentrate the chiral analyte after a previous separation (either chiral or achiral) as a type of online enrichment. Configurations that combine an achiral column for increased selectivity and trap column(s) for online enrichment are relatively common, though this type of configuration requires more columns and increases complexity. 

A. Legin, A. Rudnitskaya, D. Clapham, B. Seleznev, K. Lord, and Y. Vlasov, Electronic tongue for pharmaceutical analytics quantification of tastes and masking effects. Anal. Bioanal. Chem. 380, 36-45 (2004). 

Several major matrix types are found in marine particles and sediments. Marine organisms surround themselves with tough polymeric organic cell walls and/or with opal or calcium carbonate tests. These contrasting matrices respond differently to various analytical methods. In sediments, the remains of these organisms combine with clay minerals to form a heterogeneous mixture. In this section, the influence of these matrices on analyte quantification are discussed. 

LIF methods can be applied in-line, at-line and on-line for real-time monitoring as discussed throughout this chapter. In-line or in situ intrinsic LIF is by far more prevalent in real-time applications such as PAT as it is nondestructive and simple to deploy along with attractive analytical merits. In-line application can be accomplished by direct insertion in situ probes or flow cells. This type of monitoring is utilized for realtime analyte quantification monitoring and detection of process endpoints and faults. 

Independent Methods. In the absence of appropriate certified reference materials one may have to rely upon in-house materials that can be analyzed by independent methods (other than the candidate method). These independent methods should include a reference method and other methods that utilize different physical/chemical principles for analyte quantification. Reference methods are generally arrived at by concensus following extensive accuracy testing by a large number of laboratories. The American Society of Testing Materials (ASTM) is one of the largest compilers of reference methods. Additional information on the use of reference methods may be found in a paper by Cali and Reed (.2),... 

Although well designed LC-MS/MS assays generally outperform immunoassays due to their accuracy, sensitivity, precision, and inherent multiplexing capability, they are not free from analytical problems. Besides limitations in selectivity— isobaric analytes cannot be distinguished—sudden and unpredictable ion yield attenuations, often known as ion suppression effect, have to be considered the Achilles heel of quantitative bio-analytical mass spectrometry. Ion yield attenuation is compromising both the accuracy of an assay and its precision. It can easily lead to gross errors in analyte quantification. 

At about the same time, our laboratory has reported the development and validation of an LC tandem MS assay for as much as six TKIs simultaneously. The proposed LC-MS/MS method allows the simultaneous determination of clinically relevant ranges of concentrations for the six major TKIs currently in use imatinib, dasatinib, nilotinib, sunitinib, sorafenib, and lapatinib [122], Plasma is purified by acetonitrile protein precipitation followed by reversed-phase chromatographic separation. Analyte quantification is performed by electrospray ionization-triple quadrupole mass spectrometry by selected reaction monitoring (SRM) detection using the positive mode. This was the first broad-range LC-MS/MS assay covering the major currently in-use TKIs. 

Depending on the nature of the target, either Ag or Ab is added in excess. The excess is needed to ensure that all the analyte of interest is being complexed. A large excess facilitates a shift of equilibrium toward complex formation and in some cases can compensate for reduced affinity of the system. After incubation, the mixture is analyzed by CE. A peak of an affinity complex or a peak(s) of unbound reagent(s), resolved from each other and compared with that of calibration standards, is then used for analyte quantification. 

CE with LIF of this mixture should reveal two resolved peaks corresponding to bound and free Ab (if it still exists in solution). As in the above case, both peaks and their ratio can be used for analyte quantification. Here the higher the amount of Ab in the sample, the more Ab is displaced from the complex, and hence the higher the peak of the tracer and the lower of the complex. Besides CE-related requirements which the analyzed system should meet (see above), special attention must be paid to proper adjustment of the amount of labeled Ag and Ab added to the sample. In both extreme cases of incorrect adjustment, namely, when the amount of either Ag or Ab is too high, the results might be outside the dynamic range of the calibration curve and hence incorrect. These requirements are very typical for any mode of competitive immunoassay, including ELISA-like methods, and are discussed in detail in related books (see, e.g., Ref. 32). 

The use of MISPE for the clean-up of beef liver extracts before quantification of atrazine (5) by HPLC-UV or ELISA was evaluated by Muldoon and Stanker (entry E in Tables 15.1 and 15.2) [24]. In both cases the application of MISPE resulted in improvements in analyte quantification. In particular for the HPLC method the use of MISPE improved the accuracy and precision and lowered the limit of detection. In the case of ELISA a better accuracy was achieved in the determination although the precision was similar. With the analyte present at ppb levels, the reliability of either determination method would have been marginal without the MISPE step. 

The analytical quantification of the residual stresses on the macroscopic scale is generally based on a simple ID model [2] or the 2D plane-stress classical lamination theory [3] (CLT). 

To develop and validate a method for analytical quantification of both the substrate and the product in a fermentation process (Paper... 

Rapid quantification of products and substrates in a fermentation process is essential for process development and optimization. Most fermentation laboratories have access to HPLC equipment with possibilities to couple them to quite inexpensive diode-array-detectors, and this equipment could be used for quantitative monitoring of the process. Because HPLC can allow multi-component analyses, i.e., several analytes in the same sample can be determined virtually simultaneously, and since it is often necessary to monitor more than one substance at a time, this technique is an important tool for bioprocess monitoring. HPLC coupled to expensive MS does not represent standard equipment at fermentation laboratories. Even if mass spectrometers are available, DAD is often sufficient for quantification because product concentrations are relatively high, so the MS could be used for other issues. In paper II the goal was to develop and validate a method for analytical quantification of both the product and the substrate to enable the proper characterization of the kinetics of the process i.e., the determination of the values of substrate conversion and product formation. 

Sometimes there is some confusion over which term that should be used in characterizing a method, selectivity or specificity. Vessman [83] pointed out the differences between the two terms. Selectivity refers to a method that gives responses for a number of substances and can distinguish the analyte(s) response from all other responses. Specificity refers to a method that gives response for only one single analyte. In chromatography with UV-detectors it is unusual that a method responds only to one analyte and therefore the term selectivity is appropriate. The selectivity of the method should be evaluated by processing blank samples with and without the addition of analytes and inject them to test for interferences. The selectivity of the method is very important to enable accurate analyte quantification. 

There are no detailed recommendations for analytical quantification procedures in the field of biotechnological production of drugs, in contrast to the recommendation made by the FDA [16] for bioanalytical methods. The aim of this paper was therefore to investigate whether the latter detailed guidelines (given by the FDA for bioanalytical methods) also could be used in the field of biotechnological synthesis. Validated methods for quantification are important also in biotechnological synthesis for the proper calculation of rate coefficients. 

Where there is no appropriate standard for an analyte, quantification can be made by standard addition (spiking). In this procedure, the sample is divided into several aliquots of equal volume and a series of known but increasing amounts of the analyte standard are then added to each aliquot. The samples are then diluted to the same volume yielding a series of solutions with equal concentrations of matrix but increasing concentrations of analyte. These samples are then analyzed individually for the analyte of interest and the concentration of the unknown can then be calculated from where the regression curve of the responses versus the standard additions intercepts the abscissa (j= 0) (Figure 19). The advantage of this method is the elimination of any chemical or physical bias between the standards and samples but this is achieved at the cost of a six- or sevenfold increase in the number of determinations required for each sample. 

Relative quantitative assay is similar in approach, but generally involves the measurement of endogenously occurring analytes. Since in this case even a zero or blank calibrator will contain some amount of analyte, quantification can only be done relative to this zero level. Examples of this include immunoassays for cytokines such as interlueken-6 or certain gene expression assays (e.g., RT-PCR). 

Spectrometric methods require a prior sampling preparation containing a separation step. The separation step is necessary especially to eliminate interference. Nonspectral interferences in flame atomic absorption spectrometry can be overcome by using a calibration model.221 The model uses two independent variables for analyte quantification (the amount of the sample and the amount of analyte added) the measured absorbance is the dependent variable. To control the matrix interferences without prior knowledge of the matrix composition, it is necessary to carry out nine calibration points to obtain accurate analytical information. This confers high reliability of the analytical information for determination of trace elements in complex matrices. 

ESI-MS is often used for analyte quantification. However, a good quantification method must take into account all those parameters that during sample ionization may influence the MS signal of the analyte under investigation. The nature of the analyte, the solvent, the instrument operative parameters, the capillary tip distance from the mass spectrometer cone and the sample infusion flow rates are some examples.64 A study of the influence that some of those parameters might have on the NBDPZ signal at m/z 251 is therefore required. 

As the LOD defined by Eq. 8 defines the concentration at which one can decide whether the analyte is present, rather than quantifying the analyte concentration, another performance characteristic - the limit of quantification (LOQ) - is sometimes used [11]. Analyte quantification is generally accepted to begin at a concentration equal to 10 standard deviations of the blank. Thus, the LOQ concentration cloq, can be expressed as ... 

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