Fluorogenic labeling

Klinker CC, Bowser MT (2007) 4-Fluoro-7-nitro-2, 1, 3-benzoxadiazole as a fluorogenic labeling reagent for the in vivo analysis of amino acid neurotransmitters using online microdialysis-capillary electrophoresis. Anal Chem 79 8747-8754... 

G. Patonay and M. D. Antoine, Near-infrared fluorogenic labels New approach to an old problem, Anal. Chem. 63, 321A-327A(1991). 

H Anson-Moye, AJ Boning. A versatile fluorogenic labelling reagent for primary and secondary amines 9-fluorenylmethyl chloroformate. Anal Lett 12 25-35, 1979. 

Some cannabinoids in human urine have been analyzed by fluorogenic labeling with DNS-C1 [139]. The labeled derivatives are separated by TLC. 0.5-ng amounts of the derivatives can be detected. 

The application of fluorogenic labeling to the determination of some organophosphate insecticides has been attempted [178,179]. Fenthion (0,0-dimethyl 0-[(4-metiiylthio)-m-tolyl] phosphorothioate), Ruelene (0-2-chloro-4-ferf.-butylphenyl O-methyl methyl-phosphoramidate), GC 6506 [dimethyl p-(methylthio)phenyl phosphate] and several other compounds which yield phenols on hydrolysis have been examined. The limits of detection for some of these labeled derivatives have been reported to be in the low nanogram range. The organophosphate Proban [0,0-dimethyl 0-(p-sulphamoylphenyl) phosphorothioate] has been determined directly without hydrolysis by dansylation of the free amino group of the molecule [180]. The derivative exhibited blue fluorescence, as compared to yellow for phenol and alkylamine dansyl derivatives. 

The hydrolysis reaction is carried out in a sealed test-tube in 1 N hydrochloric acid at 150 °C for 8-16 h. The resulting solution is made basic and the mixture is then dansylated [156]. Triazines such as atrazine, simazine and propazine yield different combinations of free amines on hydrolysis, thus enabling their characterization using the fluorogenic labeling technique. The limits of detection range from 5 to 10 ng per spot. Concentrations of 0.05 ppm in food crops may be determined. 

A Critical Comparison of Pre-Column and Post-Column Fluorogenic Labeling for the HPLC Analysis of Pesticide Residues... 

Crop Extraction and Cleanup (OPA-MERC). GLYPH and AMPA were extracted and cleaned up prior to post-column fluorogenic labeling HPLC determination in exactly the same manner as for the FM0CC1 procedure with the exception that the concentrated highly acidic fractions from the 50W - X8 columns were adjusted with 10 M KOH to pH 3-8. This was necessary in order to prevent adverse shifting of the HPLC mobile phase pH and subsequent shifts in retention and deterioration of peak shape. 

The overall reproducibility of the post-column fluorogenic labeling HPLC as well as the proposed procedure for AMPA and GLYPH is illustrated by the replicate recoveries from cucumber which were measured over a two day period (Table II) ... 

Fluorogenic labelling of pesticides. The subject has been reviewed earlier by Lawrence and Frei (27). Labelling consists in replacing a proton or other atom of a pesticide with a so-called labelling compound such as dansyl chloride (V) or fluores-camine (VI). The former reacts with primary and secondary amines, phenols, some thiols and aliphatic alcohols. The latter reacts very selectively with primary amines. 

Thus, fluorogenic labelling offers an interesting approach to the analysis of pestitides on tic. It is preferable to perform the reaction on the chromatogram, rather than is solution since it is less time consuming and more practical. 

JB Shear, EB Brown, WW Webb. Multiphoton-excited fluorescence of fluorogen-labeled neurotransmitters. Anal Chem 68 1779-1783, 1996. 

Fluorescence Resonance Energy Transfer Fluorescence resonance energy transfer (FRET) real-time PGR method depends on the introduction of fluorogenic-labeled probes affected by the 5 nuclease activity of Taq DNA polymerase. The FRET method includes the TaqMan , molecular beacons scorpions and few other techniques, all of them are hybridization probes relying on fluorescence resonance energy transfer (FRET) to quantify PGR product via the generation of a fluorescent signal. 

Indole-1-carboxylic acid can be converted by treatment with a carbodiimide to the anhydride (413), which can undergo reaction with amines or the sodium salts of phenols, thiophenols, pyrroles, and indoles. For example, it reacts with the sodium salt of pyrrole-2-carbaldehyde to give the product (414) (Scheme 143) <87JOC3934>. V-Chloroformylcarbazole (415), prepared by the reaction of carbazole with phosgene, has been used effectively for the fluorogenic labeling of amino acids (Equation (118)) <90TL1455>. 

The coloured p-nitro- and 2,4-dinitrophenylhydrazones are ideal derivatives for the separation and characterization of carbonyls by paper, thin-layer and column chromatography. The oximes can easily be revealed on the thin-layer plates by spraying with solutions of copper(Il) chloride or copper(II) acetate (alcoholic) or iron(III) chloride [21,22]. For the visualization of carbonyl compounds by means of fluorogenic labeling by Schiff base formation with reagents such as dansyl-hydrazine, we refer the reader to Chapter 9. 

One of the main advantages of CE over gel electrophoresis is that the separation is monitored by online, on-column, or end-column detection. In the most frequently employed UV absorption photometric detection, a small part (less than 1mm) of the capillary serves as a detection cell. Micromolar concentrations of proteins are detectable using the low UV detection wavelength of 200-220 nm. A higher sensitivity, up to nanomolar concentrations, is achieved with fluorescence, particularly laser induced fluorescence (LIE) detection. The disadvantage of the LIE detection of proteins is the necessity for their derivatization using a fluorogenic label. The native fluorescence of proteins, mostly due to the presence of aromatic amino acids residues, tryptophan, and tyrosine, can be utilized only when low UV laser... 

Online postcolumn derivatization for LC-fluori-metric analysis of nonfluorescent or weakly fluorescent pesticides has mainly involved fluorogenic labeling of N-methyl carbamates and metabolites. The labeling reaction is a two-step process consisting of alkaline hydrolysis and posterior reaction of the hydrolysis product with o-phthalaldehyde and 2-mercaptoethanol to yield the highly fluorescent l-hydroxyethylthio-2-methylisoindole (EPA method 531.1). The detection limits for surface waters are between 20 and 30ngl . ... 

Other immunoassays No fluorogenic reaction is needed if the radiolabel or the enzyme label is replaced by a fluorogenic label, which can be measured after the bound-free separation. An example of such a label is 4-methylumbelliferone 3-acetic acid. The advantage of these fluoroimmunoassay methods (FIA) is their simplicity. However, a disadvantage is that, as a consequence of the strong background fluorescence, the sensitivity is poor. 

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