Evaluation of IMAC and Electrospray Ionization Mass Spectrometry for Recovery and Analysis of Copper-Binding Ligands in Seawater

Complexation by organic ligands dominates the speciation of iron, copper, and other bioactive trace metals in seawater, controlling their bioavailability and distribution in the marine environment. Several classes of high-affinity Fe-binding ligands (siderophores) have been identified in seawater and such compounds are known to mediate iron uptake by marine bacteria, thereby influencing biological productivity in the ocean. However, little is known about the origin, structure, or ecological role of marine Cu-binding ligands (chalcophores). Immobilized metal-ion affinity chromatography (IMAC) allows selective recovery of such compounds from seawater, while electrospray ionization mass spectrometry (ESI-MS) has been used to identify marine siderophores and to characterise Cu ligands in coastal waters. Here, we use model compounds to evaluate a Cu(II)-IMAC/ESI-MS workflow for recovery and analysis of Cu-binding ligands in seawater. One-litre samples of artificial and natural filtered seawater were spiked with model Cu(II) ligands at realistic concentrations and fractionated by IMAC. Retained compounds were eluted by acidification and detected by UV absorption. Linear plots of concentration versus UV chromatographic peak area were obtained for model synthetic and natural organic ligands at concentrations ranging from 5 to 500 nM (r²=0.9988) and 50 and 750 nM (r²=0.9899), respectively, in artificial seawater. Variable though similar results were obtained for oceanic seawater spiked with 5 nM to 1 µM of ligand (r²=0.9893). Chromatographic peak data suggests that natural UV-absorbing Cu ligands are more concentrated in nearshore than in oceanic surface waters, and that these ligands are susceptible to photolysis by artificial sunlight. Eluted IMAC fractions corresponding to UV absorbance peaks were collected and different techniques evaluated for concentration and desalting of the recovered ligands prior to analysis by ESI-MS and tandem mass spectrometry (MS/MS). MS data were used to confirm the presence of model compounds and other organic ligands in spiked samples and hence, the selective recovery and analysis of chalcophores in seawater.