Improved Method for HPLC Analysis of Polyamines, Agmatine and Aromatic Monoamines in Plant Tissue

Abstract

The high performance liquid chromatographic (HPLC) method of Flores and Galston (1982 Plant Physiol 69: 701) for the separation and quantitation of benzoylated polyamines in plant tissues has been widely adopted by other workers. However, due to previously unrecognized problems associated with the derivatization of agmatine, this important intermediate in plant polyamine metabolism cannot be quantitated using this method. Also, two polyamines, putrescine and diaminopropane, also are not well resolved using this method. A simple modification of the original HPLC procedure greatly improves the separation and quantitation of these amines, and further allows the simulation analysis of phenethylamine and tyramine, which are major monoamine constituents of tobacco and other plant tissues. We have used this modified HPLC method to characterize amine titers in suspension cultured carrot (Daucus carota L.) cells and tobacco (Nicotiana tabacum L.) leaf tissues.Polyamines are thought to play essential roles in plant growth and development (19). The PA' contents of various plant tissues have been analyzed using several ion exchange, thin-layer and high-performance liquid chromatographic procedures, each of which has its advantages and limitations.Perhaps the most widely used routine analytical method for PAs involves their separation by TLC, following prechromatographic derivatization with dansyl chloride (14). The highly fluorescent products permit excellent detection sensitivity, but quantitation of Dns-PAs on the TLC plate is cumbersome and suffers from the fact that fluorescence yield is modified by temperature, pH, and solvent polarity and the Dns derivatives are photolabile (9). Furthermore, DnsCl reacts not only with amines, but with phenolics and some alcohols, including sugars (16), and these derivatives may mask or interfere with the separation of Dns-amines on the chromatogram. Finally, the quantitation of Agm, or similar polar Dns-amine derivatives in PA mixtures, has not proved feasible using this method. DnsCl shows poor reactivity toward the '

Production of toxic pavettamine and pavettamine conjugates in the gousiekte-causing Fadogia homblei plant and its relation to the bacterial endosymbiont

“…We have developed the first method to quantify pavettamine, the toxic agent causing gousiekte, Unprotonated amines (NH 2 ) react with carbonic or sulfonic acid chlorides at alkaline pH to form amide derivatives (Slocum et al, 1989). These derivatized polyamines have good retention on reverse phase columns.…”

Relationships between polyamines, ethylene, osmoprotectants and antioxidant enzymes activities in wheat seedlings after short-term PEG- and NaCl-induced stresses

“…Then the supernatants from the two centrifugations were combined. For determination of free and conjugated PA levels, 250 ll of the supernatant was hydrolyzed with 12 mol dm -3 HCl and heated at 110°C for 18 h. After acidic hydrolysis the supernatant was evaporated and the residue was resuspended in 200 ll 10 % PCA and used for benzoylation reaction according to method of Slocum et al (1989) and Cvikrová et al (1999) with 1,7-diaminoheptane (Sigma Algrich, USA) as an internal standard. Polyamine standards (Put, Spd and Spm; Sigma, USA) were prepared similarly to plant samples.…”

Acquisition of polyamines by the obligate intracytoplasmic bacterium Rickettsia prowazekii

“…Benzoylated putrescine, benzoylated spermidine, and benzoylated spermine were separated by the method of Slocum and co-workers (22). A ternary gradient HPLC system was used with a V4 variable wavelength detector each dish, and at 48 h postinfection, the rickettsiae were isolated by N2 cavitation and Renografin.…”