RESUMO
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.
Assuntos
Ácidos/análise , Cromatografia em Gel/métodos , Compostos Orgânicos/análise , Espectrometria de Massas em Tandem/métodos , Água/química , Peso Molecular , Sensibilidade e EspecificidadeRESUMO
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.
Assuntos
Aerobiose , Anaerobiose , Pseudomonas stutzeri/metabolismo , Sideróforos/biossíntese , Regulação Bacteriana da Expressão Gênica , Pseudomonas stutzeri/genética , Pseudomonas stutzeri/crescimento & desenvolvimento , Sideróforos/química , Sideróforos/metabolismoRESUMO
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.
Assuntos
Ferricromo/análogos & derivados , Ferricromo/análise , Sideróforos/análise , Solo/análise , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas por Ionização por Electrospray , SuéciaRESUMO
Column-switching liquid chromatography followed by low-resolution ICPMS was evaluated as a tool for speciation analysis of metal-containing biomolecules. The strategy was applied on siderophores, strong iron chelators of low molecular weight (M(w) < 1500). Prior to the LC-ICPMS analysis, reductive displacement of iron by gallium was performed using ascorbate as the reducing agent to increase the sensitivity. Different experimental conditions during the exchange reaction were tested using ferrichrysin and ferrichrome for evaluation. A reaction time of 30 min and a pH of 3.9 gave an exchange yield of 27 and 83% for ferrichrysin and ferrichrome, respectively. A gradient elution profile was also developed to separate gallium-chelated siderophores on a PGC column. Detection limits for standard solutions of ferrichrysin and ferrichrome in the low-nanomolar range were obtained by monitoring the gallium-69 isotope. The combined use of LC-ICPMS and LC-ESI-MS/MS was also evaluated as a tool to identify unknown metal complexes, here siderophores, in field soil solution samples.
Assuntos
Gálio/química , Ferro/química , Metais/química , Sideróforos/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Ácido Ascórbico/análise , Ácido Ascórbico/química , Cromatografia Líquida de Alta Pressão/métodos , Gálio/análise , Ferro/análise , Estrutura Molecular , Oxirredução , Sensibilidade e Especificidade , Sideróforos/química , Espectrometria de Massas por Ionização por Electrospray/instrumentação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/instrumentação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
In this study a new approach to determine three different siderophores (ferrichrome, ferrichrysin, ferricrocin) in natural soil solutions as well as in cultures of fungi is presented. The method includes enrichment of the analytes on a short pre-column, packed with C18 material, and subsequent highly selective separation of the analytes on a capillary porous graphitic carbon (PGC) column. In contrast to normal C18 packing materials, porous graphitic carbon offers chromatographic resolution between the three very similar analytes. The selectivity of the method is enhanced even further by the electrospray ionization (ESI) mass spectrometric detection. The combination of a short pre-column and a packed capillary separation column results in a method with high sensitivity. Reported detection limits, defined as the concentration giving the signal-to-noise ratio 3:1, is 27.7 pM for ferrichrome, 46.1 pM for ferricrocin and 37.4 pM for ferrichrysin.