An improved method for analysis of polyamines in plant tissue by precolumn derivatization with o-phthalaldehyde and separation by high performance liquid chromatography

An improved high-performance liquid-chromatographic method was developed for estimation of polyamines in crude plant extracts. Polyamines were derivatized with o-phthalaldehyde and mercaptoethanol (OPT). The fluorescent derivatives were eluted from a C18 column with the dimethylcyclohexylamine-phosp...

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Bibliographic Details
Published in:Plant physiology (Bethesda) 1989-06, Vol.90 (2), p.434-439
Main Authors: Corbin, J.L. (Battelle-Kettering Research Laboratory, Yellow Springs, OH), Marsh, B.H, Peters, G.A
Format: Article
Language:English
Subjects:
ALDEHIDOS
ALDEHYDE
ALGAE
ALGUE
AMINAS
AMINE
Amines
ANALISIS DE TEJIDOS
ANALYSE DE TISSUS
AZOLLA
Biological and medical sciences
Chemical constitution
Chlorides
CHROMATOGRAPHIE
COMPOSE AROMATIQUE
COMPUESTOS AROMATICOS
CROMATOGRAFIA
Development and Growth Regulation
Elution
Fundamental and applied biological sciences. Psychology
Impurities
INVESTIGACION
Isomers
Plant physiology and development
Polyamines
Principal components analysis
Reagents
RECHERCHE
Solvents
Soybeans
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Summary:An improved high-performance liquid-chromatographic method was developed for estimation of polyamines in crude plant extracts. Polyamines were derivatized with o-phthalaldehyde and mercaptoethanol (OPT). The fluorescent derivatives were eluted from a C18 column with the dimethylcyclohexylamine-phosphate buffer derived by T. Skaaden and T. Greibrokk ([1982] J Chromatogr 247:111-122) after treatment to remove impurities in the buffer. The method had a sensitivity of 1-2 picomoles and completely resolved nine polyamines (agmatine, spermine, nor-spermidine, spermidine, 3,5-homospermidine, 4,4-homospermidine, 1,3-diaminopropane, putrescine, and cadaverine) in 12 to 14 minutes. An optional ion-exchange step was used to remove less basic amines (including amino acids) and to concentrate the crude extracts. This method was compared with benzoyl chloride derivatization. Use of the benzoyl chloride method vastly underestimated the amount of polyamine in some plant extracts, a problem not encountered with the OPT procedure. Additionally, the OPT procedure resolved two isomers of homospermidine found in Azolla caroliniana. These two isomers were not resolved with the benzoylation method. Overall, the OPT method described here requires preparation and analysis time similar to other current methods but provides greater sensitivity and selectivity
ISSN:0032-0889
1532-2548
DOI:10.1104/pp.90.2.434