title: Underivatized polyamine analysis in plant samples by ion pair LC coupled with electrospray tandem mass spectrometry.
abstract: Polyamines are key regulators of cell development and many plant responses to environmental challenges, however, their functions still remain unclear in complex interactions with other hormones and in biotic or abiotic stress. This lack of knowledge derives from the difficulties on measuring natural polyamines in plants. Here, we present a fast multiresidue method for putrescine (Put), 1,3-diaminopropane (DAP), l-ornithine, spermidine (Spd) and spermine (Spn) measurements in plant samples. Polyamine determination is based on a perchloric acid extraction followed by a simple filtration procedure without previous derivatization. Polyamines are resolved by HPLC in a C18 common column and quantified by electrospray ionization tandem mass spectrometry. (13)C(4)-putrescine and 1,7-diaminoheptane standards were added prior to sample extraction to achieve an accurate quantification in a single run. Chromatography of polyamines presents poor retention when reverse phase C18 common columns are used, because they are very polar compounds and contain several positive charges. To circumvent this problem ionic pairing technique has been used successfully with heptafluorobutyric acid (HFBA) at 1mM in the aqueous phase and 25mM in the sample. Improvement of the signal depleted by HFBA has been achieved by adding 1% of propionic acid to the aqueous and organic eluents. All together, gives a method accurate enough to determine polyamines in plants. To demonstrate the usefulness of the method it has been validated in Arabidopsis thaliana samples and polyamines have been determined in several genotypes that over express (35S::ADC2 line 3.6) or are disrupted (adc2) in the Arginine Decarboxylase2 (ADC2) gene.
authors.affiliation: Biochemistry and Plant Biotechnology Laboratory, Department of Ciencias Agrarias y del Medio Natural, Universitat Jaume I, Castellón 12071, Spain.
authors.forename: Jose
authors.initials: J
authors.lastname: Sánchez-López
authors.forename: Gemma
authors.initials: G
authors.lastname: Camañes
authors.forename: Víctor
authors.initials: V
authors.lastname: Flors
authors.forename: Cristian
authors.initials: C
authors.lastname: Vicent
authors.forename: Victoria
authors.initials: V
authors.lastname: Pastor
authors.forename: Begonya
authors.initials: B
authors.lastname: Vicedo
authors.forename: Miguel
authors.initials: M
authors.lastname: Cerezo
authors.forename: Pilar
authors.initials: P
authors.lastname: García-Agustín
mesh_terms: D017360:Arabidopsis / Q000737:chemistry* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003959:Diamines / Q000302:isolation & purification / Q000378:metabolism; D005466:Fluorocarbons / Q000302:isolation & purification / Q000378:metabolism; D007477:Ions / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism; D011073:Polyamines / Q000302:isolation & purification*; D011422:Propionates / Q000302:isolation & purification / Q000378:metabolism; D011700:Putrescine / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D003959:Diamines; D005466:Fluorocarbons; D007477:Ions; D010940:Plant Proteins; D011073:Polyamines; D011422:Propionates; C033094:perfluorobutyric acid; C015127:1,7-diaminoheptane; C029658:propionic acid; D011700:Putrescine