Fatty acids fluorescent derivatives

Acids can also be converted to fluorescent dansyl derivatives The reaction of Cg to C24 fatty acids with dansyl semipiperazide or semicadavende provides an excellent example (Fig 34) [87] Odd-numbered and unsaturated fatty acids [88] and propionic, sorbic and benzoic acid [89] can be detected in the same manner... 

Hydrazinocarbonylpropyl)-6,7-dimethoxy-l-methyl-2(177)-quinoxalinone (126) undergoes acylation by fatty acids (EtN=C=NCH2CH2CH2NMe2, H2O, pyridine, 20°C) to afford highly fluorescent derivatives that are useful for the detection and estimation of such fatty acids. ... 

HPLC has also been used for analysing fatty acid mixtures [708] and for the characterisation of fatty acids and their derivatives [709]. Fatty acids are commonly analysed on polymeric RPLC columns. Only multiple unsaturated fatty acids can be detected by UV in HPLC the others require derivatisation into UV-absorbing or fluorescing derivatives. Simultaneous determination of saturated and unsaturated fatty acids (C12-C24) by means of RPLC has been reported [710]. Derivatisation is necessary. 

DeMar Jr. J.C., Disher, R.M., and Wensel, T.G. (1992) HPLC analysis of protein-linked fatty acids using fluorescence detection of 4-(diazomethyl)-7-diethylaminocoumarin derivatives Abstract 465. Biophys. J. 61(A81). 

Colistin (COL) is a multicomponent antibiotic (polymyxins E) that is produced by strains of inverse Bacillus polymyxa. It consists of a mixture of several closely related decapeptides with a general structure composed of a cyclic heptapeptide moiety and a side chain acetylated at the N-terminus by a fatty acid. Up to 13 different components have been identified. The two main components of colistin are polymyxins El and E2 they include the same amino acids but a different fatty acid (216). A selective and sensitive HPLC method was developed for the determination of COL residues in milk and four bovine tissues (muscle, liver, kidney, and fat). The sample pretreatment consists of protein precipitation with trichloracetic acid (TCA), solid-phase purification on Cl 8 SPE cartridges, and precolumn derivatization of colistin with o-phthalaldehyde and 2-mercaptoethanol in borate buffer (pH 10.5). The last step was performed automatically, and the resulting reaction mixture was injected into a switching HPLC system including a precolumn and the reversed-phase analytical column. Fluorescence detection was used. The structural study of El and E2 derivatives was carried out by HPLC coupled with an electrospray MS. Recoveries from the preseparation procedure were higher than 60%. 

In non-viscous organic solvents the fluorescence efficiency of most SNA derivatives is comparable to that of TS as are the yields of trans to ois photoisomerization (33,3A). The double chain derivatives, MSNA and MSN, show substantially higher fluorescence yields (0.2 0.02) in non-viscous solvents with a corresponding decrease in the trans to ois isomerization efficiency (3A). In the sensitized isomerization, the MSN derivatives investigated thus far show only photoisomerization but there is an increase in the triplet lifetime by a factor of 2 however there is no change in the decay ratio of the isomerization precursor and the sum of the benzophenone sensitized isomerization efficiencies of ASA is approximately unity. When water insoluble SNA or MSNA molecules are spread as a film, either pure or in mixtures with insoluble fatty acids, at the air-water interface, it is found that the films show comparable behavior on compression to those of pure fatty acid with an indicated area per molecule of the surfactant stilbenes very close to that of a linear fatty acid (oa. 202 /molecule) (35). This suggests strongly that the TS chromophore in these molecules offers suitable molecular dimensions to pack into a crystalline-like array of linear parafin chains. 

Lloyd, J.B.F., Phenanthramidazoles as fluorescent derivatives in the analysis of fatty acids by high-performance liquid chromatography, J. Chromatogr., 189, 359, 1980. 

Used for the precolumn preparation of fluorescent derivatives of carboxylic acids reagent reacts well with fatty acids at room temperature to give intensely fluorescent esters Reference 2... 

Vesicles are ordered fluids or liquid crystals, a fact which reflects well in those photoreactivities that are particular to vesicle solutions. Fatty acid derivatives 24 and 25, for example, show low quantum yields of fluorescence ((j)f) and high quantum yields of cis-trans isomerization (< )c) as well as short fluorescence life times (tf) in both methylcyclohexane and micellar SDS solutions (Table 1). In DPPC vesicles, on the other hand, cis-trans isomerizations are cumbersome and much slower, and the fluorescence yield and lifetime rise considerably (Table 1). For those stilbene derivatives which are embedded in the middle of a fatty acid backbone, isomerization is virtually eliminated in the low-temperature or gel phase of the bilayer. The vesicle thus plays the role of stabilizing trans configurations which fit into the frozen oligomethylene chain matrix. 

Table 1 Photochemical quantum yields of fluorescence ( f), cis-trans isomerization (i c) ndfluorescence lifetimes (if) for stilbene-fatty acid derivatives in free solution, micelles and vesicles.

Kleine, and S. Petry, Analysis of lipolysis in adipocytes using a fluorescent fatty acid derivative, Biochirnie, in press. 

Free Fatty Acids and Organic Acids Free fatty acids can be readily analyzed by RPC with an acidified mobile phase and detection at 210 nm.9 However, pre-column derivatization of free fatty acids to form derivatives such as p-bromophenacyl esters, 2-nitrophenyl hydrazides, or anthryl-methylesters, can improve the chromatographic performance and detection sensitivity by UV or fluorescence detection.9 Nevertheless, GC of their methyl esters remains the more common assay method. 

The preparation and handling of the fluorescent phospholipid derivatives is cumbersome, however. Special care has to be taken to prevent degradation of the polyenoic fatty acid. Furthermore, most spectroscopic techniques require calibration to equate the spectral changes with the amount of lipid transfer. It is also important to know whether the rate of transfer of this and other fluorescent and spin-labeled phospholipids is comparable to the transfer rate of more physiological phospholipid molecules. 

In addition to parinaroyl phospholipids, pyrene fatty acid derivatives may be used. Such phospholipids have a concentration-dependent emission spectrum (Roseman and Thompson, 1980). At low concentrations of the derivative within the bilayer, the fluorescence is maximal at a wavelength below 400 nm. At higher concentrations of the derivative, the excited state monomers can associate with a ground state monomer to form a dimer complex, or eximer, in a diffusion-controlled process. The maximum emission wavelength of the eximer shifts to approximately 470 nm. The ratio of the eximer to monomer fluorescent intensity is proportional to the concentration of the probe molecules within the bilayer. 

The phenacyl esters are more readily prepared than the other derivatives. The fluorescent tag 4-bromomethyl-7-methoxycoumarin may however offer increased sensitivity. Due to the bulky nature of the fluorescent products, separation factors may be poorer and allow only positive identification of a limited number of fatty acids. 

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