Analysis of Polyamines

The first successful polyamine separations were accomplished in the mid 70s after the introduction of surface-sulfonated cation exchangers. Fig. 3-164 displays a standard chromatogram with the separation of putrescine (1,4-diaminobutane) and cadaverine (1,5-diaminopentane) as well as spermidine (A-(3-aminopropyl-1,4-diaminobutane) and 

Strength of these buffer solutions, detection cannot be carried out by means of conductivity measurements. Polyamines carry terminal NH2 groups, thus, fluorescence detection after reaction with o-phthaldialdehyde is a suitable and very sensitive detection method. 

6 mL/min detection fluorescence after reaction with o-phthaldialdehyde injection volume 20 pL solute concentrations  

Gradient Techniques in Cation Exchange Chromatography of Inorganic and Organic Cations 

Gradient elution techniques in cation exchange chromatography are less common than in anion exchange chromatography. The primary reason for this is the availability, since the late 1980 s, of separation materials that allow the simultaneous analysis of alkah- and alkaline-earth metals. Those separation materials are silica- or polymer-based weak acid cation exchangers which allow baseline-resolved separations of alkah- and alkaline-earth metals in less than 15 minutes. 

the apphcation of a gradient elution technique would not lead to a significant decrease in analysis time. This is especially true because the time required for column re-equihbration has to be taken into account. 

The first successful polyamine separations were accomplished in the mid-1970s with the introduction of surface-sulfonated cation exchangers. The separation of biogenic amins such as putrescine (1,4-diaminobutane), cadaverine (1,5-diaminopentane), spermidine (Al-(3-aminopropyl-l,4-diaminobutane), and spermine (Al,Al -bis-(3-aminopropyl)-l,4-diaminobutane) took more than 30 min. The required eluent comprised three buffer solutions that differed both in pH and in the trisodium citrate and sodium chloride concentration. Owing to the high ionic strength of these buffer solutions, detection could not be carried out by means of suppressed conductivity measurements. However, polyamines carry terminal NH2 groups and thus, fluorescence detection after reaction with 

0-phthaldialdehyde is a suitable and very sensitive detection method for this class of compounds. 

40 °C eiuent MSA (EG) gradient iinear, 3-10mmoi/L in 20 min, to 40mmoi/L in 10 min, and then to 45 mmoi/L in 15 min flow rate 0.3 mL7min detection suppressed 

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Excess of the reagent hydrolyses to a non-fluorescent residue and the reagent itself does not fluoresce. The optimum wavelength of the excitation light is 390 nm and that of the emitted light 475 nm. This regent is, however, less sensitive than Fluoropa and the derivative is unstable consequently, it must be injected onto the column immediately after formation if used in pre-column derivatization. It has been used successfully in the separation and analysis of polyamines (32), catecholamines (33) and amino acids (34). 

Teti D, Visalli M, McNair H. 2002. Analysis of polyamines as markers of (patho)physiological conditions. J Chromatogr B 781 107. 

Fluorescamine (4-phenylspiro(furan-2-(3H),r-phthalan)3,3 -dione) is also a commonly used fluorescence reagent. It reacts almost instantly and selectively with primary amines, while the excess of the reagent is hydrolyzed to a non-fluorescent product. The reagent itself is non-fluorescent. The reaction is carried out in aqueous acetone at a pH of about 8-9 and the derivatives can be chromatographed directly. The excitation and emission wavelengths are 390 nm and 475 nm respectively. Two disadvantages of the reagent are its cost and the fact the products are less stable, cannot be stored and should be injected onto the column immediately after formation. Fluorescamine has been employed in the analysis of polyamines, catecholamines and amino acids. 

The most commonly applied methods for the analysis of polyamines in erythrocytes make use of amino acid analyzers and HPLC techniques. A capillary gas chromatographic method with nitrogen-phosphorous detection was applied to the simultaneous determination of 1,3-diaminopropane, putrescine, cadaverine (Cad), spermidine (Sd), and spermine (Sp) in human erythrocytes. Blood samples, collected by venipuncture into EDTA containing Venoject tubes, were subjected to the removal of plasma by centrifugation and erythrocytes were washed three times with two volumes of 0.9% NaCl. The stability of polyamines in erythrocyte suspensions was also investigated. Quantification of polyamines was done by comparing the peak-area ratio of each analyte and its internal standard with that of the standard. The polyamine samples were eluted with 0.1 M hydrochloric acid solutions. The eluate was evaporated to dryness at 120°C under a stream of air and 200 each of acetonitrile and heptafluorobutyric anhydride were added. The isolation of derivatives... 

Slocum, R.D. Flores, H.E. Galston, A.W. Weinstein, L.H. (1989). Improved method for HPLC analysis of polyamines, agmatine and aromatic monoamines in plant tissue. Plant Physiology, Vol. 89, No. 2, pp 512-517, ISSN 0032-0889. 

Gas Chromatography-Mass Spectrometry Analysis of Polyamines in Serum Using Deuterium-Labeled Analogs as Internal Standards Acta Polyamine Res. 2 23-35 (1978) CA 88 132763c... 

Marco F, Alcazar R, Tiburcio AF, Carrasco P (201 la) Interactions between polyamines and abiotic stress pathway responses unraveled by transcriptome analysis of polyamine overproducers. OMICS 15 775-781... 

Villanueva, V. R. and Adlakha, R., Automated analysis of common basic amino acids, mono-, di-, and polyamine phenolic amines, and indoleamines in crude biological samples, Anal. Biochem., 91, 264, 1978. 

GC analyses of the pupal secretion of E. borealis have indicated the presence of vitamin E acetate and other tocopherol derivatives [49,50]. However, in tests with ants, these compounds proved to be essentially inactive, whereas the secretion itself was potently deterrent. To find and identify the active components in the pupal Epilachna borealis secretion, NMR spectroscopic studies on the fresh secretion were carried out. One and two-dimensional NMR experiments revealed that the tocopheryl acetates account for only a relatively small percentage of the beetles5 total secretion (20%), whereas the major components represented a group of previously undetected compounds. By analysis of the COSY, HSQC and HMBC spectra of the mixture, these components were shown to be esters and amides derived from three (co-l)-(2-hydroxyethylamino)alka-noic acids 44-46. HPLC analyses coupled to a mass spectrometric detector revealed that the secretion contain a highly diverse mixture of macrocyclic polyamines, the polyazamacrolides (PAMLs) 47-52 (Fig. 8). 

Several new phases applied to maltosaccharide analysis that seem to be promising have been described in the literature, for instance, a silica-phase covered with polymeric polyamine resin, and an entirely polymeric resin containing an amide function (35). In any case, partition chromatography is restricted mainly to the utilization of polar-bonded phases, as already described. Nevertheless, there have been a certain number of applications of reversed-phase chromatography that permit relatively simple separations to be achieved. Octadecyl-bonded silica phases are the most widely used, although few applications involve carbohydrate analysis. Their interest lies rather in the analysis of derivated sugars, where the selectivity increases (36). 

Three families of polymers have been used to study transfection mechanisms polyamines, polyamides, and polyvinyl type polymers. The transfection efficiencies achievable with these systems vary widely, so an in-depth analysis of each polymer family and subsequent comparison of what affects gene delivery will be discussed in this chapter. In addition to high transfection efficiency, it is important for the polymeric systems to be relatively nontoxic to cells in vitro and not to elicit an immune response in vivo. Thus, the effect of transfection parameters on cytotoxicity and immunogenicity will also be examined. 

Ilioudis, C. A., Hancock, K. S. B., Georganopoulou, D. G., Steed, J. W., Insights into supramolecular design from analysis of halide coordination geometry in a protonated polyamine matrix. New J. Chem. 2000, 24, 787-798. 

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