Characterization of copper-complexing ligands in seawater using immobilized copper(II)-ion affinity chromatography and electrospray ionization mass spectrometry

Abstract

Electrochemical studies show that copper and other bioactive trace metals are associated almost exclusively with dissolved organic ligands in surface seawater. Some of these ligands may be released by photosynthetic marine organisms to control metal uptake. However, new analytical approaches are needed to determine the chemical composition and ecological significance of these compounds. We have combined immobilized metal-ion affinity chromatography (IMAC) with electrospray ionization mass spectrometry (ESI-MS) to selectively extract and characterize copper-complexing ligands in seawater. Samples taken from British Columbia coastal waters were filtered through mixed cellulose membranes (0.45 μm) using nitrogen overpressure. Compounds with an affinity for copper were then extracted using Cu(II)-IMAC and eluted in acidified seawater. The eluant was monitored using UV absorption (255 nm), which showed that IMAC extracts from river and surface waters contained the highest concentrations of UV-absorbing organic matter. Fractions were collected and analyzed by flow-injection (FI) negative ion ESI-MS using a custom made XAD-16 column, which gave 82% recovery and a detection limit of 1.6 pmol for salicylic acid in acidified seawater. Low-molecular-weight compounds containing nitrogen were detected in the IMAC extracts of highest UV absorbance. These compounds followed an elution profile similar to the corresponding UV chromatogram. Measured masses of 265 and 259 Da are consistent with peptides containing, respectively, one thiol and two primary amino groups, characteristics associated with copper-complexing ligands detected in marine algal cultures and surface seawater using other methods. However, the absence of positive ions under experimental conditions suggests that the extracted compounds are predominantly acidic, and may therefore be hydroxamic acids or other nitrogen-containing compounds. The use of alternative resins and FI solvents to improve recovery, increase ionization efficiency, and permit structural (MS/MS) analysis of compounds extracted by IMAC is now being investigated.