Biological compounds, fluorescence analysis

Spectrophotometric Analysis. Florescamine (Fluram, Roche Diagnostics) is a nonfluorescent compound that reacts with primary and secondary amines to form fluorescent compounds. It has been used chiefly in the spectrophotometric investigation and assays of biological compounds. This investigation utilized the compound to determine concentrations of primary and secondary amines. 

Fluorescence spectrometry has become established as a routine technique in many specialized applications due to its high sensitivity. Examples of the current applications of fluorescence range from simple fluorimetric analysis for biomolecules, metal ions, and organic compounds to identification of specific DNA and/or RNA sequences in tissues. In addition, much of the current research in biochemistry, medicine, and molecular biology involves fluorescence spectroscopy of either intrinsic molecular fluorescence (from tyrosine or tryptophan residues) or exogenous fluorescent probes. 

An added feature of total fluorescence techniques is found in systems whose emission and/or excitation wavelength maxima change with the formation of a complex. Multidimensional fluorescence analysis allows the analyst to detect the differences in sensitivity arising from changes in excitation and emission maxima of the analyte. In addition, the technique finds wide application in examining the corrected fluorescence spectra of clinical compounds and their derivatives. In studies involving biological... 

A multiresidue preparation technique—MSPD—has also been applied to the analysis of CAP residues in meat samples. Two fractions were collected by elution with methylene chloride and ethyl acetate. No additional purification was necessary. Diode assay detection and fluorescence detectors were recommended for the multiresidue analysis of sulfonamides, benzimidazoles, nicarbazin, furazolidone, and CAP. The percentage recoveries and linearity of the method were evaluated. The method was linear from 50 to 250 /tg/kg of CAP. Not only do the authors recommend the MSPD multiresidue procedure for HPLC analysis, but it could be associated with several detection modes, such as immuno- or receptor assays. The MSPD technique represents a new approach in the field of biological-matrix extraction and provides a great possibility for the analysis of a wide range of compounds (20). 

Although fluorimetric analysis has been in use for a number of years in biochemistry, biology and related areas, it has had only limited use for pesticide residues and has been restricted to those compounds which exhibit a sufficiently strong intrinsic fluorescence. 

Compared to other LC detection methods, LC-MS provides better analysis for biological samples, particularly due to its excellent selectivity against interferences in the sample [29]. As reported by Kleef et al. [27], the desallyl derivative (one of the metabolites of rocuronium) could be detected in stomach fluid, but could not be quantitated by fluorescence detection due the existence of fluorescent endogenous substances in the sample. In addition, LC-MS requires no prior sample derivatization as do other detection methods [27, 29]. Detection of analytes in biological samples using LC-MS with electrospray ionization is suitable for the ionization of polar compounds such as rocuronium bromide [30]. 

The spectral and topographic analysis of fluorescence associated with biologically active compounds (1-11) is of great interest in the study of cell metabolism (12,13,14) and cell-to-cell interactions (15,16,17). The dynamics of intracellular metabolism (]8,19,20,21") are better unraveled if transient shifts from steady state are"triggered via microinjections of metabolites, which requires instrumental arrangements allowing micro-manipulatory techniques (22,23,24). 

Analyses of tropane alkaloids are mainly carried out by GC and HPLC and to a lesser extent by CE. This review describes recent applications developed for the analysis of this class of compounds in plant materials and biological matrices. Of course, mass spectrometry is generally used as the detection technique because of its high sensitivity and selectivity, but other techniques such as UV, fluorescence, flame ionization detection, nuclear magnetic resonance, among others have also been investigated. Finally, desorption electrospray ionization mass spectrometry is reported as a new interesting detection technique for the rapid analysis of samples without any sample preparation. 

The use fluorescence detection in the chromatographic analysis of biological matrices has found widespread applicability because of its inherent sensitivity and specificity (B25, H5, H7-H9, K23, K29, K32, Yl, Y2). Since fluorescence is more sensitive than UV spectroscopy, it can be used advantageously to analyze for naturally fluorescent compounds in the presence of nonfluorescing, UV-absorbing compounds. In such cases, fluorometric detection is more sensitive while at the same time possible interferences are eliminated. 

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