Fluorescence chemiluminescence detection

While compendial standards are available for some monographed article impurities, it may be difficult at times to obtain pure standards of impurities. Manufacturers of pharmaceuticals function as a potential source for obtaining reference standards of impurities, which may be synthesis precursors, process intermediates, or degradation products. The characterization and evaluation of these impurities reference standards should be constant with their intended use. In many cases, analytical procedures are developed and validated, where the response of an impurity is compared to that of the new drug substance itself. Response factor evaluation of impurities at the chosen detection wavelength is necessary to determine if a correction factor is needed (when the responses differ). Potentiometric detection, fluorescence/ chemiluminescence detection, and refractive index detection are some examples of detection modes available for compounds that may not be suitable for UV detection. 

Concerns over safe handling of radioactive materials and issues around the cost and disposal of low level radioactive waste has stimulated the development of nonradiometric products and technologies with the aim of replacing radioactive tracers in research and medical diagnosis (25). However, for many of the appHcations described, radioactive tracer technology is expected to continue to be widely used because of its sensitivity and specificity when compared with colorimetric, fluorescent, or chemiluminescent detection methods. 

Table V. Comparison of Limits of Detection (LOD) for Dansyl and NDA Derivatives Employing Fluorescence and Chemiluminescence Detection for RP-HPLC...

Fluorescence detection is usually more sensitive than absorption detection, but the number of naturally fluorescent compounds is limited. Pre- or postcolumn derivatization can also be applied for this type of detection. Chemiluminescence detection is the most sensitive method for some fluorescent compounds. 

Fluorescence detection relies on the visualization of a secondary antibody that has been labeled with a fluorophore such as fluorescein (FITC), Texas Red, Tetramethyl rhodamine (TRITC), or R-phycoerythrin. Although this method of detection has a reduced sensitivity of twofold to fourfold compared to chemiluminescence detection, it presents a tenfold greater linear dynamic range, thus providing better linearity and better quantiflcation within the detection limits. Since secondary antibodies can be labeled with fluor-ophores of distinct colors, multiplexing (simultaneous detection of several antigens) of the same blot is feasible. 

Alternate derivatization techniques have also been used. These include dansylhydrazine with fluorescence or chemiluminescence detection of the hydrazone (e.g., Nondek et al., 1992 Rodler et al., 1993), 3-methyl-2-... 

Finally, we must point out that nowadays the widely extended RILAs are basically used for MIP characterization purposes. In a general way, the use of RILAs has been reduced drastically in the last few years. The literature shows that they have been replaced by other tracers with fluorescence, chemiluminescence or absorbance detection often combined with labeled enzymes. Simplicity in their use, easy access to these techniques, and the lack of radioactive compounds are the main reasons for such substitution. 

Method of detection Fluorescence Chemiluminescence Fluorescence Change in pH... 

Fan and Zhang determined acetylcholine and choline in rat brain tissue by a fluorescence immunoassay method, making use of immobilized enzymes and chemiluminescence detection [50]. Tissue was homogenized with a 10 fold volume of 0.6 M HC104, the homogenates were kept on ice for 30 min, and then centrifuged at 2000 G for 20 min. The pellets were... 

Sulfur contents were determined by non-dispersive X-ray fluorescence spectrometry (ASTM D 4294-83). The nitrogen content was analyzed by mieans of oxidative combustion and chemiluminescence detection (ASTM D 4629-86),... 

Flavor compounds, detection of 938 Flavyhum salts 1029 Floating peroxyl radical theory 889 Fluorescence/chemiluminescence techniques, in measurement of antioxidant efficiency 853, 854... 

Nondek L, Milofsky RE, Birks JW. 1991. Determination of carbonyl compounds in air by HPLC using on-line analyzed microcartridges, fluorescence and chemiluminescence detection. Chromatographia 32 33-39. 

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