LC–MS analysis of low molecular weight organic acids derived from root exudation

Abstract

A sensitive method for quantification of citric, fumaric, malic, malonic, oxalic, trans aconitic, and succinic acid in soil- and root-related samples is presented. The method is based on a novel, fast, and simple esterification procedure and subsequent analysis via liquid chromatography-mass spectrometry. Derivatization comprises in situ generation of HCl, which catalyzes the Fischer esterification with benzyl alcohol. As a key advance, the esterification with the aromate allows reversed-phase separation and improves electrospray ionization efficiency. The method provided procedural detection limits of 1 nM for citric, 47 nM for fumaric, 10 nM for malic, 10 nM for malonic, 16 nM for oxalic, 15 nM for succinic, and 2 nM for aconitic acid utilizing 500 μL of liquid sample. The working range was 3 nM to 10 μM for citric acid, 158 nM to 10 μM for fumaric acid, 34 nM to 10 μM for malic acid, 33 nM to 10 μM for malonic acid, 53 nM to 10 μM for oxalic acid, 48 nM to 10 μM for succinic acid, and 6 nM to 10 μM for aconitic acid. Quantification of the analytes in soil-related samples was performed via external calibration of the entire procedure utilizing (13)C-labeled oxalic and citric acid as internal standards. The robustness of the method was tested with soil extracts and samples from hydroponic experiments. The latter concerned the regulation of phosphorus solubilization via plant root exudation of citric, malic, and oxalic acid.

Profiling of secondary metabolites in root exudates of Arabidopsis thaliana

“…The nutrient solution from the hydroponic cultivation system contains inorganic macronutrients in the low millimolar concentration range, whereas root exudates are expected to occur at a 3-6 orders of magnitude lower concentration (Jaitz et al, 2011). Hence, nontargeted LC/MS-based profiling of root exudates requires an extensive sample preparation which allows for an at least 1000-fold enrichment of exuded components from the nutrient solution and an efficient depletion of bulk components such as inorganic salts at once.…”

Bioavailability of zinc and phosphorus in calcareous soils as affected by citrate exudation

“…Feodora) seeds were germinated in quartz sand and were transplanted after 5 days to 50 L containers filled with a continuously aerated nutrient solution. The composition of the nutrient solution was based on Jaitz et al (2011). The pH of the nutrient solution was adjusted to 7.3 (±0.2).…”

Bacterially Induced Weathering of Ultramafic Rock and Its Implications for Phytoextraction

“…Carboxylic acids were separated by reversed-phase liquid chromatography on a C 18 column with 5-m particle size (GraceSmart, RP18, and 2.1-by 150-mm column from Grace Davison Discovery Sciences, Deerfield, IL, USA) and analyzed by liquid chromatography-electrospray ionization-time of flight mass spectrometry (LC-ESI-TOF MS) (Agilent Technologies, Palo Alto, CA, USA) as described by Jaitz et al (19). Quantification of selected phenolic compounds (caffeic acid, catechin, p-coumaric acid, 2.5-dihydroxybenzoic acid, epicatechin, ferulic acid, gallic acid, 4-hydroxy-3-methoxycinnamidaldeyhde, resveratrol [3,5,4=-trihydroxy-trans-stilbene], sinapic acid, syringic acid, and vanillic acid) was performed via LC-tandem MS (MS-MS) in negative ionization mode on a 6410 triple-quadrupole mass spectrometer from Agilent Technologies (Palo Alto, CA, USA) equipped with an ESI interface (20).…”