Quantification ofethylenediamine-N,N-bis(hydroxysulfophenylacetic) acidregioisomers and structural characterisation of its relatedpolycondensation products by porous graphitic carbonhigh-performance liquid chromatography coupled to electrospraytandem mass spectrometry

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 oneof the promising chelates for the treatment of chlorotic plants. Industrial synthesis of EDDHSA/Fe leadsto relevant amounts of o,o-EDDHSA condensation products (o,o-EDDHSAcps) and other secondary prod-ucts that might have important relevance from the agronomic point of view. However, their chemicalstructures have remained unknown to date. Analysis of iron complexes by ion-pair reversed-phasechromatography, coupled with electrospray tandem mass spectrometry revealed the presence of themeso-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 num-bers of stereoisomers and the poor efficiency of the ion-pair reversed-phase separation method. Analternative 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 detectionof EDDHSA stereo/regioisomers, EDDHSAcps, and low-molecular-weight by-products. This extensivePGC–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-weightby-products. PGC separation coupled to a triple quadrupole ESI-MS detector allowed characterisationof free ligands using collision-induced dissociation experiments in positive and negative ionisationmode, providing comparative evaluation of EDDHSAcps in three commercial samples. For detection, thePGC–HPLC–ESI-MS/MS is the best method according to the limit of quantification and limit of detec-tion (picomolar and sub-picomolar detection, respectively) for determination of meso-EDDHSA andrac-o,o-EDDHSA. Synthesis, purification and quantification of o,o-EDDHSA and o,p-EDDHSA by1H-nuclearmagnetic resonance are also reported