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