Multicomponent assays

Multicomponent analysis in immunoassay has recently become an important technique and in principle this type of assay should be possible in FIA. The labels for multicomponent analysis must be chosen to have excitation and emission characteristics which enable them to be determined independently in the same solution. The assay may take the form where the excitation of the two overlap and the emission bands are separated, and in this case it may be possible to excite the molecules simultaneously and split the emission to two detectors giving simultaneous measurements. Alternatively, the labels may be measured independently. In certain cases derivative or synchronous spectra may be used to further resolve the labels. 

Multicomponent FIA methods may be carried out in solution or they may be solid-phase. In the case of solid-phase assays, the antibodies may be immobilized randomly on the solid phase or in sections which may be viewed selectively. 

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LC-MS/MS has dramatically changed the way bionalysis is conducted. Accurate and precise quantitation in the pg ml scale is nowadays possible however one has to be aware of certain issues which are specific to mass spectrometric detection such as matrix effects and metabolite crosstalk. With the current growing interest in the analysis of endogenous biomarkers in biological matrices, quantitative bioanalysis with MS has certainly the potential to contribute further in this field with the development of multicomponent assays. Modern triple quadrupole instruments have the feature to use very short dwell times (5-10 ms), allowing the simultaneous determination of more than 100 analytes within the timescale of an HPLC peak. Due to the selectivity of the MS detection the various analytes... 

H.M. Heise, R. Marbach, Th. Koschinsky, F.A. Gries, Multicomponent Assay for Blood Substrates in Human Plasma by Mid-infrared Spectroscopy and its Evaluation for Chnical Analysis , AppZ. Spectrosc., 48,85-95 (1994). 

Buryak, A. Pozdnoukhov, A. Severin, K. Pattern-based sensing of nucleotides in aqueous solution with a multicomponent indicator displacement assay. Chem. Commun. 2007, 2366-2368. 

An ultraviolet spectroscopic method was presented, and used for the assay of procaine and nitrofural in a multicomponent collagen sponge without prior separation of the drugs [33]. Crushed Collagen Sponge (0.1 g) was dissolved in 70 mL of 1 mM HCl, and heated for ten minutes. The solution was cooled, diluted to volume, mixed, filtered, whereupon the first 20 mL was discarded. The absorbance of the analyte solution was then measured at 290 and 373 nm (against 1 mM HCl) for procaine and nitrofural, respectively. 

Revalidation of Minor Changes. A minor change to the drug product may require revalidation of only a single validation parameter. For example, a change in the supplier of a cherry flavor for a multicomponent cough syrup would require a revalidation of specificity in the HPLC assay procedure to confirm the lack of interference from the new flavor source. Only if there... 

An antennal-specific aldehyde oxidase (AOX) of M. sexta (MsexAOX) was the next identified pheromone-degrading enzyme (Rybczynski el al., 1989). The activity of MsexAOX was visualized on non-denaturing PAGE, and was shown to be antennal specific but present in sensilla of both male and female antennae. MsexAOX was observed as a dimer with a combined estimated molecular mass of 295 kDa. M. sexta uses a multicomponent pheromone consisting exclusively of aldehydes including bombykal (Starratt el al., 1979 Tumlinson el al., 1989, 1994) MsexAOX was shown to degrade bombykal to its carboxylic acid. Both TLC and spectrophotometric assays were established and a variety of substrates and inhibitors were characterized. Making adjustments for the concentrations and volumes within a sensillum lumen, the in vivo half-life of pheromone was estimated at 0.6 msec in the presence of this enzyme (Rybczynski el al., 1989). 

Crowther and Henion have reported the SFC assay of polar drugs on packed columns using mass spectrometric detection [10]. Their method was shown to be suitable for a multicomponent mixtures containing nonpolar and polar analytes. They mentioned that one of the advantages of SFC versus HPLC was the faster column reequilibration time. In this method, silica, amino, nitrile, and diol packed columns (20 cm x 2.1 mm ID) were used. Cocaine, codeine, caffeine, methocarbamol, phenylbutazone and ox-yphenbutazone were separated on one or more of these columns and mass spectra were obtained. 

Different reversed phase [195,239,240], mixed mode (ion exchange and reversed phase) SPE cartridges [173,218] and online SPE column [193, 238] have been also reported for samples preparation and extraction. Some of these assays combined both PP and SPE in order to achieve an extensive sample cleanup [193, 195, 238-240], Likewise SPE, LLE provides cleaner plasma extracts than PP. Nevertheless, LLE procedure does not always provide satisfactory results with regard to extraction recovery and selectivity, especially with polar analytes and particularly in the case of multicomponent analysis such as in drug-metabolism studies, where analytes polarity varies widely. This issue was addressed by Zweigenbaum J and Henion J [235] and extraction solvent optimization, using isoamyl alcohol, to achieve acceptable extraction selectivity and recovery for polar analytes has been discussed. 

Techniques in clinical analysis have undergone many advances in the last few decades. The basic needs in clinical chemistry are unambiguous analyte-specific assays that provide both identification of sample components and their concentration levels. The importance of this is self-evident, since most substances analyzed are part of a multicomponent biological fluid. Advances in enzyme and immunochemical assay techniques provide ideal systems for component... 

The same group has also repotted a multicomponent Hantzsch protocol which provided access to polycyclic indenoheterocycles 78 utilizing a variety of aromatic and heterocyclic amines (Scheme 19) [45]. Some of the compounds disclosed showed apoptosis-inducing activity in a human T-cell leukemia cell line similar to etoposide. One of the most potent analogs was compound 79 (Scheme 19) which showed greater than 30% induction of apoptosis at 25 pM, as assessed in a flow cytometric aimexin V/propidium iodide assay in Jurkat cells. 

The first series of compounds assayed directly by CD detection were the morphine alkaloids. They were supported in aqueous solutions, in a chiral cholesteric liquid crystal solvent, and mixed in pellet form with solid KBr. ° Contrary to expectations, the homogeneous aqueous solution medium gave the best selectivity among 10 related opiates and the most quantitative results. The pH-dependence of phenol substituted analogs, which in some instances caused the sign of the CD signal to invert, enhanced the selectivity. Heroin was assayed both directly and as the morphine hydroly-sate.f Direct multicomponent analyses were made for prepared mixtures of morphine, codeine, thebaine, noscapine, and opium extracts. ... 

In conclusion, it can be stated that spectroscopic techniques will further dominate the analytical tools of the future with respect to qualitative and quantitative assays. This is because of their speed and the enormous information content of the spectra, especially in the infrared, and the fact that reagent-free multicomponent methodologies are available. The widespread diffuse reflection technique certainly has to compete with others in the laboratory and at the production site. However, for the study of bulk and dispersed systems, it will often be the method of choice. There are additional developments concerned with dedicated instruments and user-friendly interfaces, in which che-mometrics play an important role. It is hoped that the sophisticated algorithms presented in the literature will... 

Sulfacetamide, A benzoyl sulfanilamide and sulfathiazole have been determined in pharmaceutical preparations by measuring absorbance of the mixture in 0.1 N hydrochloric acid at 220,235 and 280 nm respectively (42). Madsen et al. (43,44) have performed computer analysis of the multicomponent UV spectra of sulfonamides. The errors in concentration determined from the spectra between 240 and 272 nm are lower when the spectra are analysed by a linear-squares method considering the data over the whole wavelength range compared with the determination using the data at a single wave length. The method has been applied to the assay of sulfacetamide sodium eye-drops. 

In contrast, with its intrinsically higher selectivity, tandem mass spectrometry offers a much more effective and practical approach for multicomponent quantitation. This translates into faster method development and, ultimately, rapid and reliable multicomponent quantitation. The routine application of multicomponent quantitation to 5 or 10 compounds in a cassette can be developed in 1 or 2 days, offering typical quantitation range of 5 to 5000 ng/mL. The variability of the assay procedure ranges from 10 to 20%, which is less than the typical intersubject pharmacokinetic variability of 25 to 30%. 

An added complexity, but one that may better predict therapeutic activity, is the testing of drugs in assays with different contexts (i.e., basal stimulation). Testing in vivo can further produce therapeutic model systems. Certain multicomponent disease conditions can be adequately modeled only in vivo. The ultimate model is the human in the clinical situation. Translational medicine with noninvasive imaging techniques and biomarkers now are able to furnish valuable information that can be used in the initial discovery process to produce better-defined drugs. 

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