Anal. Chem. 2004, 76, 2618-2622
Fingerprinting Metal-Containing Biomolecules after Reductive Displacement of Iron by Gallium and Subsequent Column-Switched LC-ICPMS Analysis Applied on Siderophores My Moberg,† Eva M. Nilsson,† Sara J. M. Holmstro 1 m,‡ Ulla S. Lundstro 1 m,‡ Jean Pettersson,*,† and † Karin E. Markides
Department of Analytical Chemistry, Uppsala University, P.O. Box 599, SE-751 24 Uppsala, Sweden, and Department of Natural and Environmental Sciences, Mid Sweden University, SE-851 70 Sundsvall, Sweden
Column-switching liquid chromatography followed by lowresolution 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 (Mw < 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. Many analytical problems concern identification and quantification of compounds containing both metal/metals and an organic part in different matrixes. For instance, organometallic compounds of arsenic, selenium, and tin are frequently analyzed in environmental studies. In the same way, many biomolecules, such as transport proteins and enzymes, form complexes with metals. Yet another field of interest is the analysis of metal complexes that are biosynthesized by plants and animals as a response to metal stress or deficiency. Siderophores are excreted by microbes, fungi, and some plants during iron-limited conditions to form watersoluble complexes with iron(III). The formation of these complexes facilitates the acquisition of iron into the organism. Most siderophores can be classified as either hydroxamates or phenolates-catecholates and have a molecular weight of less than 1500.1 The identification of specific siderophores and determination of their concentrations are of special interest in the understanding * Corresponding author: (e-mail)
[email protected]. † Uppsala University. ‡ Mid Sweden University. (1) Payne, S. M. Methods Enzymol. 1994, 235, 329-344.
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of plant nutrition. Siderophores are also believed to contribute to the weathering of minerals, and hence, analytical methods to study these compounds in field soil solution and culture samples are of great importance.2,3 Due to the complexity of the natural samples, unknown siderophores are not easily detected by, for example, LC-ESIMS.4 The reason is that other compounds, also present in the sample, will give rise to a number of additional peaks in the obtained mass spectra. Also, the low concentrations of siderophores, typically in the low-nanomolar range, found in natural field soil solutions makes the identification with LC-ESI-MS even more troublesome. Instead, selective monitoring of iron in known and unknown siderophores could be performed with argon plasma spectrometry. The main isotope of iron, 56Fe, is, however, subjected to severe spectral interferences from mainly argon polyions under normal inductively coupled plasma (ICP) conditions; hence, a resolution of over 2500 would be needed to separate 56Fe from 40Ar16O+ in ICPMS. This resolution is only attainable by a sector-field (SF) instrument.5 Quadrupole (Q) instruments equipped with a hexapole6 or octapole7 reaction or collision cell can, on the other hand, almost entirely eliminate the argon interferences on the iron isotopes. Combined with a reaction or collision cell, the ICP-QMS can give low ppt detection limits for iron in bulk analysis,7,8 which are about the same or slightly higher than what is attainable with an ICP-SFMS instrument.5 The ICPSFMS and ICP-QMS instruments supplied with a reaction or collision cell are, however, still rather rarely occurring. Medium ppt detection limits for 56Fe can be obtained in pure standards under cold plasma condition with time-of-flight (TOF) ICPMS9 or ICP-QMS10 instruments. This is a factor of ∼2 orders of magnitude (2) Kalinowski, B. E.; Liermann, L. J.; Givens, S.; Brantley, S. L. Chem. Geol. 2000, 169, 357-370. (3) Wattueau, F.; Berthelin, J. Eur. J. Soil Biol. 1994, 30, 1-9. (4) McCormack, P.; Worsfold, P. J.; Gledhill, M. Anal. Chem. 2003, 75, 26472652. (5) Jakubowski, N.; Moens, L.; Vanhaecke, F. Spectrochim. Acta, Part B 1998, 53, 1739-1763. (6) Feldmann, I.; Jakubowski, N.; Thomas, C.; Stuewer, D. Fresenius J. Anal. Chem. 1999, 365, 422-428. (7) Leonhard, P.; Pepelnik, R.; Prange, A.; Yamada, N.; Yamada, T. J. Anal. At. Spectrom. 2002, 17, 189-196. (8) O’Brien, S. E.; Acon, B. W.; Boulyga, S. F.; Becker, J. S.; Dietze, H.-J.; Montaser, A. J Anal. At. Spectrom. 2003, 18, 230-238. 10.1021/ac0355000 CCC: $27.50
© 2004 American Chemical Society Published on Web 04/07/2004
lower than what is obtainable by monitoring the 56Fe isotope under normal plasma conditions by ICP-QMS,10 but still too high for trace siderophore analysis. Problems with increased matrix effects and a less robust plasma are, however, also inherent with the cold plasma technique, which makes it unsuitable for coupling to a reversed-phase LC system. The less interfered 54Fe and 57Fe isotopes can be measured under normal plasma conditions,11 but a direct aspiration detection limit of 0.7 ppb for 57Fe,12 which is of the same magnitude as that for ICP atomic emission spectrometry,13 is not sufficient for trace siderophore analysis. As is obvious, alternative approaches to analyze siderophores are great needed. In addition to iron, other trivalent metal ions such as aluminum, chromium, gallium, and indium also form complexes with siderophores.14 Gallium(III) has an ionic radius similar to trivalent iron and forms octahedral complexes with oxygen ligands. Since siderophores chelate Fe(III) but not Fe(II) and since no divalent oxidation state of gallium is known, exchange of iron by gallium can be performed in a reducing environment. In the case of ferrichrome and other hydroxamate-type siderophores, iron has been exchanged by gallium to a larger or lesser extent by a 10fold excess of gallium with15 or without16 a reducing agent present. Recently, an enormous excess of gallium (1000 ppm added) was used to displace iron by gallium to be able to identify the presence of a siderophore in a seawater sample by LC-ESI-MS.4 Gallium, in contrast to iron, is well suited for analysis by ICPMS. The 69Ga isotope, which has a natural abundance of 60%, is not subjected to spectral interferences from argon. The detection limit for gallium in bulk analysis with an ICP-TOFMS instrument is at the low ppt level,9 1 and 3 orders of magnitude worse compared to what can be obtained with ICP-QMS17 and ICPSFMS,18 respectively, but still low enough to enable trace analysis of gallium. Hence, an analytical method based on the exchange of iron by gallium and subsequent ICP-TOFMS detection would offer both a sensitive and a selective approach to analyze the natural occurrence of siderophores. The objectives of this study were to investigate the applicability of ICP-TOFMS following reversed-phase LC separation of different gallium-chelated siderophores monitoring the 69Ga isotope and to determine optimal conditions for reductive exchange of iron by gallium in siderophores using ascorbate as reducing agent. Development of the LC method was based on results from a previous report,19 which included enrichment of the siderophores (9) Tian, X.; Emteborg, H.; Adams, F. C. J. Anal. At. Spectrom. 1999, 14, 18071814. (10) Huang, L.-S.; Lin, K.-C. Spectrochim. Acta, Part B 2001, 56, 123-128. (11) Stuhne-Sekalec, L.; Xu, S. X.; Parkes, J. G.; Olivieri, N. F.; Templeton, D. M. Anal. Biochem. 1992, 205, 278-284. (12) Almeida, C. M.; Vasconcelos, M. T. S. D. Anal. Chim. Acta 2002, 463, 165-175. (13) Seubert, A. Trends Anal. Chem. 2001, 20, 274-287. (14) Raymond, K. N.; Mu ¨ ller, G.; Matzanke, B. F. In Complexation of Iron by Siderophores: A Review of Their Solution and Structural Chemistry and Biological Function; Boschke, F. L. Ed.; Springer: New York, 1984; Vol. 123, pp 49-102. (15) Emery, T. Biochemistry 1986, 25, 4629-4633. (16) Emery, T.; Hoffer, P. B. J. Nucl. Med. 1980, 21, 935-939. (17) Horlick, G.; Montaser, A. Inductively Coupled Plasma Mass Spectrometry; Wiley-VCH Inc.: New York, 1998. (18) Sohrin, Y.; Iwamoto, S.; Akiyama, S.; Fujita, T.; Kugii, T.; Obata, H.; Nakayama, E.; Goda, S.; Fujishima, Y.; Hasegawa, H.; Ueda, K.; Matsui, M. Anal. Chim. Acta 1998, 363, 11-19. (19) Moberg, M.; Holmstro ¨m, S. J. M.; Lundstro ¨m, U. S.; Markides, K. E. J. Chromatogr., A 2003, 1020, 91-98.
Table 1. Parameter Settings for the Reductive Exchange Experiments levels in experimental design reaction time
10 min
20 min
30 min
pH
3.9
4.9
5.8
on a precolumn and subsequent separation on a porous graphitic carbon (PGC) column. Different conditions for reductive exchange were evaluated using the hydroxamate siderophores ferrichrysin and ferrichrome produced by fungi. The developed setup in combination with LC coupled to ESI-MS and ESI-MS/MS was finally utilized to identify siderophores in a natural field soil solution sample. EXPERIMENTAL SECTION Chemicals. Methanol and acetonitrile of LiChrosolv grade together with formic acid, ascorbate, orthophosphoric acid (85%), sodium dihydrogen phosphate, disodium hydrogen phosphate, and iron(III) nitrate of analytical grade were purchased from Merck (Darmstadt, Germany). Desferrichrome, desferrioxamine mesylate, disodium hydrogen citrate, and gallium(III) nitrate were obtained from Sigma-Aldrich (GmbH, Munich, Germany). Ammonium formate of analytical grade was purchased from BDH Laboratory Supplies (Poole, U.K.). Desferrichrysin and desferricoprogen were bought from Biophore Research Products (EMC Microcollections GmbH, Tu¨bingen, Germany). All solutions were prepared with water purified with a MilliQ-system (Millipore, Bedford, MA). Reductive Exchange Experiments. Experiments according to a full three-level factorial design were performed to establish the influence of pH and reaction time on the exchange yield of iron by gallium; the different levels are displayed in Table 1. Two experiments were performed for each experimental condition, and external calibration curves were utilized to determine the different exchange yields. The solutions were prepared by mixing ferrichrome and ferrichrysin with ascorbate and gallium nitrate in 50 mM phosphate buffer at different pH. Two milliliters of each solution was prepared with a final concentration of ∼150 nM of each ferrisiderophore, 120 µM ascorbate, and 3 µM gallium(III). The prepared solution was shaken on a Vortex for 1 min and then immediately inserted into a sonication bath, the sonication was stopped according to the reaction time of that specific experiment, and finally 300 µL of the solution was injected onto the chromatographic system. Exchange yields were calculated using Microsoft Excel (Version 97, Microsoft), and response surface models were calculated with Minitab (Version 12.1, Minitab Inc.). Preparation of Standard Solutions and Natural Samples. Standard solutions were prepared by mixing desferrisiderophores with gallium nitrate in 30 mM ammonium formate and 20 mM formic acid. Soil solution was sampled from the organic mor layer of a podzolized soil at the island Alno¨ (62°24′N, 17°30′E) in the vicinity of Sundsvall, Sweden. The site is forested with Scots pine (Pinus sylvestris) and Norway spruce (Picea abies) ∼25 years old, and the field layer was dominated by lingonberries (Vaccinium vitis-idea), blueberries (Vaccinium myrtillus), and heather (Calluna vulgaris). The sample was prepared according to the procedure Analytical Chemistry, Vol. 76, No. 9, May 1, 2004
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described in a previous report.19 Prior to injection on the columnswitched LC system, reductive exchange of the natural sample was accomplished in phosphate buffer (50 mM, pH 3.9) by addition of ascorbate and gallium(III). The final concentration was 120 µM with respect to ascorbate and 60 µM with respect to gallium(III). In another experiment, the natural sample was spiked with ferrioxamine, ferrichrysin, ferrichrome, and iron-bound coprogen prior to the reductive exchange. In both experiments with natural samples, the reaction time was 20 min. Chromatography. The chromatographic setup was based on the method described in a previous report.19 A buffer containing 6 mM ammonium formate and 4 mM formic acid in MilliQ water was prepared. Three different mobile phases were obtained by mixing the buffer with organic modifier. Mobile phase A was a mixture of buffer and methanol, 99:1 v/v; mobile phases B and C were mixtures of buffer and acetonitrile, 95:5 and 1:1 v/v, respectively. Two six-port injection valves (Valco Instruments Co. Inc., Houston, TX) were used in the experimental setup. In one valve, a 300-µL injection loop was fitted, and in the other the precolumn, a HyPurity Aquastar column (10 mm × 2.1 mm, 5 µm, Thermo Hypersil-Keystone, Cheshire, U.K.), and the separation column, a Hypercarb column (100 mm × 2.1 mm, 5 µm, Thermo Hypersil-Keystone), were fitted. The former column was a C18 column with incorporated hydrophilic groups and the latter a PGC-based column. In the loading position, the precolumn was pumped with mobile phase A using a Jasco PU-980 pump (Jasco, Tokyo, Japan) at a flow rate of 300 µL/min and the separation column was pumped with a mixture of mobile phases B and C using a Rheos 2000 pump (Flux Instruments AG, Basel, Switzerland) at a flow rate of 150 µL/min. When the valve was switched to unload sample, both the pre- and separation columns were pumped with the Rheos pump at 150 µL/min. The separation of the siderophores was accomplished with isocratic elution with 20% mobile phase C during the experiments according to the experimental design described earlier. In all other applications, elution was accomplished using a gradient from 0 to 25% C in 1 min, 25% C for another 9 min, 25-95% C in 10 min, and finally 95% C for 10 min. The separation column was connected to the ICPMS via a 175-mm-long and 0.13-µm-i.d. PEEK tubing connected to a 46mm-long and 0.5-µm-i.d. Teflon tubing fitting to the nebulizer. Mass Spectrometry. Detection of 69Ga was performed using a Renaissance ICP-TOFMS (Leco, St. Jospeh, MI)9 equipped with a PFA MicroFlow ST nebulizer (Elemental Scientific Inc., Omaha, NE) and a GlassExpansion water-jacketed cyclonic spray chamber (Glass Expansion SARL, Romainmoˆtier, Switzerland) kept at 5 or 11 °C. Typical ICP conditions: plasma gas flow rate 14.9 L/min, auxiliary gas flow rate 1.0 L/min, nebulizer gas flow rate 0.65 L/min, and forward power 1.23 kW. The gas flow rates, forward power, and torch position were optimized while pumping a 50 ppb elemental gallium standard solution in 22% mobile phase C at a flow rate of 150 µL/min. Two percent of oxygen gas was added to the sample aerosol to prevent carbon deposits on the sampler cone. The obtained chromatographic peaks were integrated using the peak-fitting module Origin 6.0 (Microcal Software Inc. Northampton, MA).
as field soil solutions, column-switching LC offers preconcentration and cleanup of the sample on-line. Here, the combination of efficient preconcentration using a short solid-phase extraction column and subsequent sufficient separation of the analytes on PGC column coupled to ICPMS detection provided a simultaneous sensitive and selective element-specific method. Experiments were performed to determine the retention of the analytes, citrate, and ascorbate on the precolumn. Low molecular weight organic acids, like citrate, are present in most soil systems in the low micromolar concentration range (0-50 µM)20 and may affect the retention of the analytes on the precolumn, and thus, standard solutions of gallium desferrichrome with or without addition of citrate were analyzed. It was found that addition of 77 or 500 µM citrate did not affect the retention of the analyte and that no citrate was injected onto the separation column if the valve was switched 5 min after injection. The retention of the reducing agent, i.e., ascorbate, on the precolumn was also determined using ESI-MS. It has previously been shown that chemical redox reactions of PGC can change the chromatographic behavior of the packing material.21,22 Hence it is important to prevent the reducing agent from entering the separation column. The ESIMS experiment showed no retention of ascorbate on the precolumn, and thus, ascorbate will not be injected onto the PGC column using the column-switching technique. In our previous paper, we used isocratic elution of ferrichrome, ferrichrysin, and ferricrocin from the PGC column.19 In this study, one aim was to develop a more general separation method that can be applied to essentially all kinds of siderophores; thus, different gradient profiles were evaluated. Figure 1 shows the separation of gallium chelates of desferrioxamine (1), desferrichrysin (2), desferrichrome (3), and desferricoprogen (4) with the final profile, i.e., 0-25% C in 1 min, 25% C for 9 min, 25-95% C in 10 min, and finally 95% C for 10 min, with ICPMS detection. External calibration curves for standard solutions containing gallium-chelated ferrichrysin and ferrichrome were established; see results in Table 2. The calculated concentrations of the different gallium desferrisiderophores in the standard solutions anticipate 100% binding of the siderophore ligands to gallium. For
RESULTS AND DISCUSSION Column-Switching Chromatography Coupled to ICPMS. For complex samples that contain a variety of components, such
(20) Strobel, B. W. Geoderma 2001, 99, 169-198. (21) To ¨rnkvist, A.; Markides, K. E.; Nyholm, L. Analyst 2003, 128, 844-848. (22) Shibukawa, M.; Unno, A.; Miura, T.; Nagoya, A.; Oguma, K. Anal. Chem. 2003, 75, 2775-2783.
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Figure 1. Analysis of gallium-chelated siderophores with the column-switched LC-ICPMS system. Elution order: (1) Ga-desferrioxamine, (2) Ga-desferrichrysin, (3) Ga-desferrichrome, and (4) Ga-coprogen. Sample: 51.2 nM Ga-desferrioxamine, 102.5 nM Ga-desferrichrysin, 11.3 nM Ga-desferrichrome, and 37.6 nM Gacoprogen, Vinj ) 300 µL. Conditions: mobile phase A loads sample onto the precolumn (HyPurity Aquastar, 10 mm × 2.1 mm) at 300 µL/min; a gradient from 0 to 25% C in 1 min, 25% C for 9 min, 2595% C in 10 min, and 95% C for 10 min at 150 µL/min separates the analytes on a Hypercarb column (100 mm × 2.1 mm); columnswitching time 5 min.
Table 2. Calibration Results, Limits of Detection (LOD), 3:1 S/N, and R Valuesa compound
concn range (nM)
LOD (nM)
R value
Ga-desferrichrysin Ga-desferrichrome
8.7-343.5 9.1-299.4
1 0.8
0.999 1.000
a Results from the injection of five standard solutions containing mixtures of gallium-chelated desferrichrysin and desferrichrome in Milli-Q water.
iron-bound siderophores at a pH above 2, no appreciable dissociation into free ligand and free iron is exhibited,14 and since reported stability constants for iron and gallium are both high (compare for desferrioxamine B log β ) 30.99 for iron and 28.17 for gallium),23 this assumption seems justified. As can be seen, detection limits in the low-nanomolar range were obtained for the analytes, which makes the method promising for analysis of siderophores in natural samples. The detection limit in real samples will of course also be dependent on the exchange yield of iron by gallium for the siderophore at hand and also the level of natural sample dilution during the reductive exchange experiment. Other features that may affect the detection limit of a specific siderophore are the percentage acetonitrile in the chromatographic peak, since this may have an effect on the ICPMS response, and also the width of the obtained chromatographic peak. However, with our instrumental setup, we did not experience any pronounced difference in response due to variation in acetonitrile content. Furthermore, the proposed method is qualitative or semiquantitative rather than quantitative, since no distinction between coeluting siderophores can be made using exclusively LC-ICPMS. Optimization of Reductive Exchange of Iron by Gallium. Hydroxamate and catecholate siderophores have low affinities for iron(II), since reduction of iron makes the complex unstable with respect to protonation and dissociation. Consequently, iron can be replaced by gallium in a reducing environment.15 An enormous excess of gallium has also been shown to replace iron to some extent.4,16 This method is questioned though,16 since it will only work for siderophores that bind iron rather weakly. Different parameters will affect the reductive exchange yield of iron by gallium. For instance, the concentration of reducing agent and the amount of gallium added to the sample may have an effect on the exchange yield. The concentrations of these additives should be considered in relation to the total siderophore content in the sample, and since this will not be known a priori, we chose not to try different concentrations. Instead, we always added a large excess of both reducing agent and gallium. The reaction time and the pH can also affect the exchange yield.15 Experiments according to an experimental design (see the Experimental Section) were performed to study the influence of these two parameters in detail. Multiple linear regression was utilized to make models from the experimental results using coded settings between -1 and +1 for the different parameters. Models containing main, first-order interaction and quadratic terms were calculated. The obtained R2 values were 81.4% for ferrichrysin and 94.1% for ferrichrome. Figure 2 shows how the exchange yield (23) Kiss, T.; Farkas, E. J. Inclusion Phenom. 1998, 32, 385-403.
Figure 2. Reductive exchange yields of iron by gallium for ferrichrysin and ferrichrome at different experimental conditions.
Figure 3. Chemical structures of ferrichrysin and ferrichrome.24,25
varies with applied experimental conditions. The contour plots show that the highest exchange yield is obtained using a low pH and a long reaction time for both analytes, at pH 3.9, and at a reaction time of 30 min, the exchange yield is 27 and 83% for ferrichrysin and ferrichrome (with corresponding standard deviation estimated to 8 and 10%, respectively). It could be argued that the experimental domain should be extended since the highest exchange yield is obtained at the highest level tested for reaction time and the lowest level tested for pH. These extremes were, however, chosen with some thought. The pH range was restricted to what you normally find in field soil solutions and the maximum reaction time was reasoned from the total time of analysis of an LC-ICPMS run, and consequently, we chose not to extend the experimental domain further. The different exchange yields obtained for ferrichrysin and ferrichrome are somewhat surprising, since the two analytes are very similar in structure (see Figure 3). A rather large difference in exchange rate had been reported earlier for hydroxamate siderophores belonging to the ferrichrome family, e.g., for ferrichrome A and ferrichrome.15 Also, other constituents in the sample matrix may effect the exchange yield of iron by gallium. In the pH range 2-6, citrate is known to form stable polymers with gallium.26 Thus, if citrate is present in the sample, the exchange yield may be affected. To investigate this possible phenomenon, exchange experiments were (24) Norrestam, R.; Stensland, B.; Bra¨nde´n, C. I. J. Mol. Biol. 1975, 99, 501506. (25) Llina´s, M.; Klein, M. P.; Neilands, J. B. J. Mol. Biol. 1972, 68, 265-284.
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the experimental design also showed that no significant matrix effects were exhibited for ferrichrysin, implying that the sensitivity of the developed method is approximately the same for galliumchelated siderophores in natural samples as in standard solutions.
Figure 4. Analysis of a coniferous soil solution sample from Alno¨, Sweden, using the column-switched system. Elution order: (1) Gadesferrioxamine and (2) Ga-desferricrocin. Reductive exchange conditions: reaction time 20 min and pH 3.9. Chromatographic conditions according to description in Figure 1.
performed with and without citrate in the sample. The reaction time was set to 20 min and the pH was 4.9. No significant difference in exchange yield was, however, observed for these experiments, comparing 9 and 10% for ferrichrysin, and two measures of 18% for ferrichrome (the standard deviation was estimated to 3 and 2%, respectively). Application on a Natural Sample. To investigate the potential of the developed method to make fingerprints of the siderophore content in natural samples with different origins, a reductive exchange experiment was performed on a field soil solution sample; see the Experimental Section for details. Figure 4 shows the resulting 69Ga trace from this experiment, and as can be seen, two peaks were clearly recognized. The same sample, with the same sample preparation, was also analyzed using a Q TRAP instrument (MDS Sciex, Concord, ON, Canada) to further investigate the origin of these peaks. The chromatographic system was coupled to the instrument, and LC-ESI-MS experiments were performed to determine the corresponding precursor ions for the obtained siderophore peaks. Enhanced product ion scan experiments were subsequently conducted on the identified precursor ions, and fragmentation patterns were established. The obtained fragmentation spectra were compared with results from direct infusion experiments on different gallium-chelated siderophores. From these comparisons, it was clear that the first-eluting peak corresponded to Ga-desferrioxamine and the second was identified as Ga-desferricrocin. The similar retention behavior of these two peaks compared to corresponding gallium-chelated standards also validated these results. The natural sample was also spiked with ferrioxamine, ferrichrysin, and iron-bound coprogen to investigate whether the sample matrix had any influence on the chromatographic elution profile, but no difference was observed. Comparing the result from the spiked sample with results from (26) Glickson, J. D.; Pitner, T. P.; Webb, J.; Gams, R. A. J. Am. Chem. Soc. 1975, 97, 1679-1683. (27) Maher, J. P. Annu. Rep. Prog. Chem, Sect. A. 2000, 96, 45-57. (28) Łobin´ski, R.; Szpunar, J. Anal. Chim. Acta 1999, 400, 321-332.
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CONCLUSIONS The utility of ICP-TOFMS followed by column-switching LC has been recognized as an appealing tool for analysis of metalcontaining biomolecules. The hyphenation of LC and ICPMS provides high selectivity in the identification of both known and unknown metal-containing compounds. In this study, we have chosen to work with iron-containing siderophores, but the applied methodology could easily be adapted to other applications with iron complexes. The strategy to reductively exchange iron by gallium has proven valuable due to the low detection limits obtained for gallium compared to iron with LC-ICP-TOFMS. The use of gallium is today mainly restricted to two areas: the semiconductor industry and in medicine for organ scanning and as an anticancer agent.27 In these fields, as well as in studies of the increasing levels of gallium in the environment and what effect it has on different biological species, there may also be a growing need for sensitive analytical methods where gallium is separated from other elements or where gallium-containing species are separated from each other. Moreover, we have exemplified the strength of combining two different ionization techniques to identify unknown metal-containing biomolecules in complex samples. With ICPMS, a specific element, in this case gallium, could be detected in a compound, and ESI-MS together with ESIMS/MS could then be utilized to further characterize the compound. The combination of hard and soft ionization techniques has mainly been applied to applications within the traditional speciation analysis, for instance, in selenium speciation.28 It is our hope and confirmed belief that the application of combined LCICPMS and LC-ESI-MS/MS analysis will be increasingly extended into new fields of speciation analysis. ACKNOWLEDGMENT We thank Thermo Hypersil-Keystone (UK) for kindly providing Hypercarb columns and MDS Sciex for making the Q TRAP available for ESI-MS and ESI-MS/MS analysis. Financial support from the Swedish Research Council project 621-2002-3918, The Swedish Foundation of Strategic Research, and the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning is also acknowledged.
Received for review December 18, 2003. Accepted March 2, 2004. AC0355000
