Analysis of low molecular mass organic acids in natural waters by ion exclusion chromatography tandem mass spectrometry.

A sensitive and selective method for the analysis of aliphatic low molecular mass organic acids (LMMOAs) in natural waters is presented. The method is based on separation with ion exclusion chromatography and detection with electrospray ionization tandem mass spectrometry (LC-MS/MS). The extra selectivity gained by applying MS/MS allows for a minimum of sample preparation and the use of a sub-optimal mobile phase regarding chromatographic resolution. Instead the mobile phase, comprising aqueous formic acid with methanol as organic modifier, was mainly optimized for maximum sensitivity and long term MS stability. Detection limits for malonic, fumaric, maleic, succinic, citraconic, glutaric, malic, alpha-ketoglutaric, tartaric, shikimic, trans-aconitic, cis-aconitic, isocitric and citric acid were in the range 1-50 nM, while the detection limits for pyruvic, oxalic and lactic acid were around 250 nM for an injection volume of 100 microL. Due to their metal-chelating properties, these LMMOAs are all considered to affect the bioavailability of metals and to be involved in soil forming processes. It is thus of interest to be able to monitor their presence in natural waters, and the method developed within this work was successfully applied for the analysis of LMMOAs in soil solution and stream water samples.

Siderophore production by Pseudomonas stutzeri under aerobic and anaerobic conditions.

The siderophore production of the facultative anaerobe Pseudomonas stutzeri, strain CCUG 36651, grown under both aerobic and anaerobic conditions, was investigated by liquid chromatography and mass spectrometry. The bacterial strain has been isolated at a 626-m depth at the Aspö Hard Rock Laboratory, where experiments concerning the geological disposal of nuclear waste are performed. In bacterial culture extracts, the iron in the siderophore complexes was replaced by gallium to facilitate siderophore identification by mass spectrometry. P. stutzeri was shown to produce ferrioxamine E (nocardamine) as the main siderophore together with ferrioxamine G and two cyclic ferrioxamines having molecular masses 14 and 28 atomic mass units lower than that of ferrioxamine E, suggested to be ferrioxamine D(2) and ferrioxamine X(1), respectively. In contrast, no siderophores were observed from anaerobically grown P. stutzeri. None of the siderophores produced by aerobically grown P. stutzeri were found in anaerobic natural water samples from the Aspö Hard Rock Laboratory.

Quantification of hydroxamate siderophores in soil solutions of podzolic soil profiles in Sweden.

Concentrations up to 2 and 12 nM of the hydroxamate siderophores ferrichrome and ferricrocin, respectively, were identified in soil solutions of podzolic forest soils at four sites in both northern and southern Sweden. No ferrichrysin was detected. As with the dissolved organic carbon and low molecular mass organic acids, the highest concentrations of the siderophores were found in the upper layers i.e. the mor layer, the eluvial and upper illuvial horizons. At the southern sites, the concentrations of ferrichrome and ferricrocin were both of similar magnitude and did not differ between the two sites. In contrast, soil solutions at the two northern sites contained more ferricrocin than ferrichrome; the ferricrocin concentrations were also higher at the northern sites than at the southern sites. Analyses were performed by high performance liquid chromatography with a porous graphitic carbon column on which ferrichrome, ferricrocin and ferrichrysin were separated. Detection by electrospray ionization mass spectrometry (ESI-MS) combined with on-line sample pre-concentration, by means of column-switching, enabled detection limits of 0.1-0.2 nM for ferrichrome, ferrichrysin and ferricrocin. The structural identities of the siderophores were further verified by MS/MS fragmentation. Fragmentation of ferrichrome, ferricrocin and ferrichrysin occurred mainly via peptide cleavage. The most intense fragments were typified by the loss of one of the three iron(III) chelating hydroxamate residues, i.e N(5)-acyl-N(5)-hydroxy ornithine.