title: Quantification of ethylenediamine-N,N'-bis(hydroxysulfophenylacetic) acid regioisomers and structural characterisation of its related polycondensation products by porous graphitic carbon high-performance liquid chromatography coupled to electrospray tandem mass spectrometry.
abstract: Among the commercial ethylenediamine-N,N'-bis(2-hydroxy)phenylacetic acid/iron(III) derivatives, ethylenediamine-N,N'-bis(2-hydroxy-5-sulphophenylacetic) acid/iron(III) (EDDHSA/Fe) represents one of the promising chelates for the treatment of chlorotic plants. Industrial synthesis of EDDHSA/Fe leads to relevant amounts of o,o-EDDHSA condensation products (o,o-EDDHSAcps) and other secondary products that might have important relevance from the agronomic point of view. However, their chemical structures have remained unknown to date. Analysis of iron complexes by ion-pair reversed-phase chromatography, coupled with electrospray tandem mass spectrometry revealed the presence of the meso-o,o-EDDHSA/Fe, rac-o,o-EDDHSA/Fe, o,p-EDDHSA/Fe regioisomers, the hydroxyl derivative of o,o-EDDHSA/Fe, and the three main EDDHSA condensation products chelating the iron(III) (EDDHSAcps/nFe). However, the chromatographic peaks of EDDHSAcps/Fe are not well resolved due to the large numbers of stereoisomers and the poor efficiency of the ion-pair reversed-phase separation method. An alternative chromatographic method is based on porous graphitic carbon (PGC) separation after pre-column decomplexation of the chelates with trifluoracetic acid, which was developed to allow detection of EDDHSA stereo/regioisomers, EDDHSAcps, and low-molecular-weight by-products. This extensive PGC-HPLC-ESI-MS/MS investigation provides quantitative determination of meso-o,o-EDDHSA, rac-o,o-EDDHSA and o,p-EDDHSA, in addition to characterisation of EDDHSAcps and the low-molecular-weight by-products. PGC separation coupled to a triple quadrupole ESI-MS detector allowed characterisation of free ligands using collision-induced dissociation experiments in positive and negative ionisation mode, providing comparative evaluation of EDDHSAcps in three commercial samples. For detection, the PGC-HPLC-ESI-MS/MS is the best method according to the limit of quantification and limit of detection (picomolar and sub-picomolar detection, respectively) for determination of meso-EDDHSA and rac-o,o-EDDHSA. Synthesis, purification and quantification of o,o-EDDHSA and o,p-EDDHSA by (1)H-nuclear magnetic resonance are also reported.
authors.affiliation: Valagro SpA, Via Cagliari 1, 66041 Atessa, Italy. Electronic address: a.biasone@valagro.com.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Biasone
authors.forename: Giusto
authors.initials: G
authors.lastname: Cianci
authors.forename: Donata
authors.initials: D
authors.lastname: Di Tommaso
authors.forename: Alberto
authors.initials: A
authors.lastname: Piaggesi
authors.forename: Nicola
authors.initials: N
authors.lastname: D'Alessandro
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006108:Graphite / Q000737:chemistry*; D007477:Ions / Q000737:chemistry; D010648:Phenylacetates / Q000032:analysis* / Q000737:chemistry*; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization; D013237:Stereoisomerism; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D007477:Ions; C501683:N,N'-ethylenediamine-di-(2-hidroxy-5-sulfophenylacetic) acid; D010648:Phenylacetates; D006108:Graphite
keywords: EDDHSA; EDDHSA condensation products; ESI-MS/MS; Iron complexes; Porous graphitic carbon