title: Simultaneous measurement of proline and related compounds in oak leaves by high-performance ligand-exchange chromatography and electrospray ionization mass spectrometry for environmental stress studies.
abstract: A mass spectrometer was coupled to high-performance ligand-exchange liquid chromatography (HPLEC) for simultaneous analysis of stress associated solutes such as proline, hydroxyproline, methylproline, glycine betaine and trigonelline extracted from leaves of drought stressed oaks and an internal standard namely N-acetylproline. Methanol/chloroform/water extracts were analyzed using an Aminex HPX-87C column and specifically quantified by the positive ion mode of an electrospray ionisation-mass spectrometry (ESI-MS) in single ion monitoring (SIM) mode. The recovery of N-acetyl proline added to oak leaf extracts ranged from 85.2 to 122.1% for an intra-day study. Standard calibration curves showed good linearity in the measured range from 0.3125 to 10micromolL(-1) with the lowest correlation coefficient of 0.99961 for trigonelline. The advantages of this alternative procedure, compared to previously published methods using fluorescence or amperometric detections, are the simultaneous and direct detection of osmoprotectants in a single chromatographic run, a minimal sample preparation, a good specificity and reduced limits of quantification, ranging from 0.1 to 0.6micromolL(-1). Fifty-six days of water deficit exposure resulted in increased foliar free proline levels (2.4-fold, P<0.001, 155micromolg(-1) FW) and glycine betaine contents (2.5-fold, P<0.05, 175micromolg(-1) FW) of drought stressed oak compared to control.
authors.affiliation: Public Research Center-Gabriel Lippmann, Department of Environment and Agrobiotechnologies (EVA), 41 rue du Brill, L-4422 Belvaux, Luxembourg.
authors.forename: Mouhssin
authors.initials: M
authors.lastname: Oufir
authors.forename: Nadine
authors.initials: N
authors.lastname: Schulz
authors.forename: Patan Shaik
authors.initials: PS
authors.lastname: Sha Vallikhan
authors.forename: Eva
authors.initials: E
authors.lastname: Wilhelm
authors.forename: Kornel
authors.initials: K
authors.lastname: Burg
authors.forename: Jean-Francois
authors.initials: JF
authors.lastname: Hausman
authors.forename: Lucien
authors.initials: L
authors.lastname: Hoffmann
authors.forename: Cedric
authors.initials: C
authors.lastname: Guignard
mesh_terms: D000470:Alkaloids / Q000032:analysis; D001622:Betaine / Q000032:analysis; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D055864:Droughts; D016014:Linear Models; D018515:Plant Leaves / Q000737:chemistry*; D011392:Proline / Q000031:analogs & derivatives* / Q000032:analysis*; D029963:Quercus / Q000737:chemistry*; D012015:Reference Standards; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013312:Stress, Physiological
chemical_list: D000470:Alkaloids; C009560:trigonelline; D001622:Betaine; D011392:Proline