Anal Bioanal Chem (2006) 385: 821–833 DOI 10.1007/s00216-006-0456-8
ORIGINA L PA PER
Helle Rüsz Hansen . Spiros A. Pergantis
Mass spectrometric identification and characterization of antimony complexes with ribose-containing biomolecules and an RNA oligomer Received: 3 February 2006 / Revised: 27 March 2006 / Accepted: 28 March 2006 / Published online: 13 June 2006 # Springer-Verlag 2006
Abstract Mass spectrometric techniques have been used to study the interaction of inorganic Sb(V) with biomolecules containing a ribose or deoxyribose moiety. Electrospray (ES) mass spectra of reaction mixtures containing inorganic Sb(V) and one of several biomolecules (adenosine, cytidine, guanosine, uridine, adenosine-5′-monophosphate, adenosine-3′,5′-cyclic monophosphate, ribose, or 2′-deoxyadenosine) afforded high-mass antimony-containing ions corresponding to Sb(V)–biomolecule complexes of stoichiometry 1:1, 1:2, or 1:3. The complexes were characterized by collision-induced dissociation (CID) tandem mass spectrometry (MS) using ion-trap multistage MS. The CID results revealed that Sb(V) binds to the ribose or deoxyribose moiety. Structures are proposed for the Sb– biomolecule complexes. Analysis of the reaction mixtures by reversed-phase chromatography coupled on-line to either inductively coupled plasma (ICP) MS or ES–MS showed that in solution Sb(V) forms complexes with all the analyzed biomolecules with vicinal cis hydroxyl groups. Evidence (from size-exclusion chromatography ICP–MS and direct infusion ES–MS) of complexation of Sb(V) with an RNA oligomer, but not with a DNA oligomer, supports the suggestion that the presence of vicinal cis hydroxyl groups is critical for complexation to occur. This is the first direct evidence of complexation of Sb(V) with RNA. Results obtained by studying the effect of changing reaction conditions, i.e. pH, reaction time, and Sb/ biomolecule molar ratio, on the extent of Sb–biomolecule formation suggest the reaction may be of physiological Electronic Supplementary Material Supplementary material is available for this article at http://dx.doi.org/10.1007/s00216-0060456-8 and is accessible for authorized users. H. R. Hansen . S. A. Pergantis (*) Department of Chemistry, Environmental Chemical Processes Laboratory, University of Crete, P.O. Box 2208, Voutes, 71003 Heraklion, Greece e-mail:
[email protected] Tel.: +30-2810-545084 Fax: +30-2810-545001
importance. Selected reaction monitoring (SRM) and precursor-ion-scanning tandem MS were investigated to determine their potential to detect trace levels of the Sb– biomolecule complexes in biological samples. Application of SRM MS–MS in combination with high-performance liquid chromatography enabled successful detection of an Sb–adenosine complex that had been spiked into a complex biological matrix (liver homogenate). Keywords Antimony . RNA . Ribonucleoside . Chromatography . Electrospray . ICP–MS
Introduction Some antimony compounds are used for treatment of leishmaniasis [1]. At elevated levels, however, antimony is acutely toxic [2]; some of its species are reported to cause DNA damage [3] and Sb2O3 has been assigned by the International Agency for Research on Cancer to the group of substances which are suspected human carcinogens [4]. Despite the high toxicity and therapeutic use of antimony, little is known about its biochemical mode of action, even though several studies have attempted to investigate this. More specifically, antimony has been shown to form, in vitro, complexes with several organic ligands and biomolecules, e.g. citric acid [5], phthalic acid [6], trypanothione [7], glutathionine [8], and N-methyl-D-glucamine [9]. It is also interesting to note that only a few organoantimony compounds have been identified in environmental and in biological samples [10–13]. This may be partially attributed to the element’s low natural abundance and the lack of sufficiently sensitive and selective detection techniques suitable for antimony speciation analysis. Another difficulty with this type of analysis is the requirement for analytical conditions which preserve possible antimony–biomolecule complexes during extraction and analysis. Complexation between chemicals used for antimony speciation must be avoided; e.g. complexation with acetic acid, a commonly used chromatographic reagent, has been reported [14]. Insufficient effort has yet
822
been made to overcome these challenges, however, as demonstrated by the limited progress made toward development of methods suitable for antimony speciation in biological samples. Both nucleosides and nucleotides contain metal-binding sites. In particular, metals of the first transition row have high affinity for the nitrogen of the nucleobase [15]. Boron, a semi metal of group 13 of the periodic table, on the other hand, has extremely high affinity for the cis-diols on the ribose moiety [16]. Inorganic antimony in the+III and+V oxidation states has also been shown to form complexes with the ribose moiety. Kluefers and Mayer reported, after use of NMR spectroscopy, that Sb(III) binds to guanosine at both the 2′ and 3′ hydroxyl positions of the ribose moiety to form a 1:2 Sb–guanosine complex [17]. In a more recent paper, Demicheli et al. suggested, after use of NMR and circular dichroism (CD), that Sb(V) binds to the ribose moiety of adenosine and adenosine monophosphate (AMP) at the 2′ hydroxyl position only [18]. Titrations of the nucleosides with Sb(V), then CD, indicated the formation of a 1:2 Sb–adenosine complex, and uncharacterized higher-ratio complexes. The involvement of the 2′ OH group as the binding site for Sb(V) was recently confirmed by the use of NMR in a study on the complexation of Sb(V) with guanosine 5′-monophosphate (GMP) and guanosine 5′-diphospho-D-mannose (GDP-mannose) [19]. This study reported the dominant presence of 1:1 Sb–biomolecule complexes compared with the 1:2 complexes. The objectives of this study were: 1. to evaluate whether complexes of Sb with some biomolecules were detectable by ES; 2. to characterize the binding sites by MS and MS–MS, if possible; 3. to determine which classes of biomolecules (nucleosides or modified nucleosides with mono hydroxyl or cis diol groups, e.g. adenosine, cytidine, guanosine, uridine, ribose, 2′-deoxyribose, 2′-deoxyadenosine, adenosine-5′-monophosphate, adenosine-3′,5′-cyclic monophosphate (Fig. 1a), and, finally, an RNA and a DNA oligomer) bind to inorganic Sb(V); and 4. to evaluate the possibility of using MS methods for Sb-speciation in a biological system. Characterization of Sb–biomolecule complexes provides information about the fundamental interactions of inorganic Sb(V) with nucleosides and relevant analogs. More specifically, electrospray tandem mass spectrometry (ES–MS–MS) both with and without high-performance liquid chromatography (HPLC) was used for characterization of complexes of inorganic Sb with biomolecules. HPLC on-line with inductively coupled plasma (ICP)–MS was also used to obtain complimentary structural and mass-balance information for the antimony biomolecule reactions.
Experimental Materials Adenosine, 2′-deoxyadenosine monohydrate, adenosine3′,5′-cyclic monophosphate (3′,5′-CAMP), and formic acid were all purchased from Fluka (Buchs, Switzerland). Cytidine and ribose were purchased from Fluka, China, and guanosine from Fluka, Italy. Uridine, adenosine-5′-monophoshpate disodium salt (AMP), adenine, ammonium hydroxide (25 %), and ammonium acetate (>98 %) were all purchased from Fluka (Seelze, Germany), 2′-deoxyribose, acetic acid (glacial), and acetonitrile (>99.8 %) were obtained from Merck (Darmstadt, Germany). Potassium hexahydroxoantimonate (99 %) was purchased from Riedel–de Haen (Seelze, Germany), and ammonium formate (99.995 %) from Sigma Aldrich (Steinheim, Germany). HPLCpurified RNA oligonucleotide 5′-AGUCCAGUUGAGU CU-3′ (Mr=4753.9 Da) was obtained from Microsynth (Balgach, Switzerland) and HPLC-purified DNA oligonucleotide 5′-TATAG-3′ (Mr=1502.0 Da) from Sigma– Genosys (Steinheim, Germany). Deionized water (18 MΩ cm) was obtained from an Ultra Clear Basic water-purification system from SG, Germany. Sample preparation A stock solution of inorganic Sb(V) (5 mmol L−1 or 605 μg Sb mL−1) was prepared by dissolving potassium hexahydroxoantimonate in deionized water. Stock solutions containing individual biomolecules were prepared at concentrations from 4–18 mmol L−1 by dissolving the appropriate compound in deionized water. Dissolution of adenine required addition of a small amount of acid (formic acid) and dissolution of guanosine required either acid or base (aqueous ammonia). All stock solutions were sufficiently stable when stored at 4 °C; only the guanosine and adenine stock solutions were prepared fresh before each reaction. To obtain results that could be conveniently compared with those of Demicheli et al. [18], and on the basis of preliminary experiments, in-vitro reactions between inorganic Sb(V) and individual biomolecules were conducted at a molar ratio of 1:2 as follows: an aqueous solution containing a single bio-compound was added to an inorganic Sb(V)-containing solution to give a final concentration of 0.82 mmol L−1 Sb(V) (100 mg Sb L−1) and 1.64 mmol L−1 biomolecule. Finally, the pH was adjusted to the desired value, typically pH 5 unless otherwise stated, by dropwise addition of a dilute solution of formic acid (0.005 %).
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Because of the low pH of the stock solutions of guanosine or adenine, their reactions with Sb(V) in water did not require further acidification. Similarly, antimony-free biomolecule solutions and biomoleculefree antimony solutions were prepared as controls. All equipment used in the work with RNA and DNA oligomers was sterilized before use. Glassware, including nanospray needles, was baked in an oven at 180 °C overnight. Diethyl pyrocarbonate (DEPC) 0.05 % was added to de-ionized water and left overnight before being autoclaved to remove any trace of DEPC. The sterilized water was used throughout the experiment. Pipette tips and other plastic-ware were also autoclaved before use. Gloves were worn at all times during these experiments. Working solutions of 0.82 mmol L−1 hexahydroxy antimonate (Sb(V)), potassium carbonate, RNA, or DNA were prepared in sterilized water. Sb(V) and the RNA or DNA were added together in a ratio of 1:1 and left at room temperature for at least 20 min before analysis unless otherwise indicated. To obtain RNA and DNA control samples with the same amount of potassium, potassium carbonate was added to these reactions instead of Sb(V). The pH of the reactions was either kept neutral (pH 7) or acidified to pH 5 by the addition of formic acid. A fresh swine liver sample was obtained from a local retailer and a small amount of the tissue was homogenized in water (1:1). The Sb–adenosine reaction solution (330 mg Sb L−1, 0.2 mL) was spiked into 2.0 mL crude liver homogenate to give a final concentration of 30 mg Sb L−1. The sample was sonicated (15 min) before centrifugation (Universal II, Hettich, Germany) until full separation of the supernatant from the residue. The supernatant was filtered (syringe filter 0.45 μm×25 mm) and diluted with water (1:1) to give a final concentration of 15 mg Sb L−1 before analysis.
auxiliary gas and argon as collision gas (1.0 mTorr). The heated capillary was maintained at 350 °C. Because of our particular instrument setup HPLC was used solely in combination with the triple-quadrupole instrument and not the IT. The injection volume was 20 μL and a piece of PEEK tubing was used to connect the outlet of the HPLC column directly to the ES–MS. Inductively coupled plasma MS (ICP–MS) was performed with a Thermo Electron X series ICP–MS, equipped with an impact bead spray chamber and a pneumatic nebulizer. PEEK tubing was used to connect the outlet of the HPLC column directly to the inlet of the ICP nebulizer. The HPLC system consisted of a Marathon Series IV pump with a Rheodyne 6-port sample-injection valve (loop volume 20 or 35 μL). A tube supplying an internal standard (20 μg Ge or In L−1) at a flow rate of 0.5 mL min−1 was mixed with the column eluent via a T-junction. Signals at m/z 121, 123 (corresponding to 121 Sb and 123Sb) and 74 or 115 (corresponding to 74Ge or 115 In) were measured. In addition, m/z 31 (31P) and 39 39 ( K) were recorded in the analysis of RNA and DNA oligomers. Samples were diluted with deionized water (and sterilized when required) typically to contain 10– 500 μg Sb L−1 and subsequently injected without filtration. Concentrations of antimony species were determined from a five-point calibration curve of Sb(V) (0, 5, 10, 25, 50 μg Sb L−1) by integration of peak area using PlasmaLab Software (Thermo Electron). All mass spectrometers were operated with unit mass resolution and their mass accuracy, typically ±0.2, was checked daily by running a sodium trifluoroacetate tuning solution. Chromatography
Instrumentation Ion trap mass spectrometry (IT-MS) experiments were performed with a Thermo Electron LCQ Advantage mass spectrometer equipped with an electrospray (ES) source (for analysis of Sb–biomolecule interaction) or a nanospray (NS) source (for analysis of Sb–DNA and Sb–RNA interaction). Samples were infused directly into the ES source at 3 μL min−1, using the built-in syringe pump. Data were typically collected in full-scan mode, with a range of m/z from 200 to 2000, in both negative and positiveionization modes. The sheath and auxiliary gas was nitrogen. Collision-induced dissociation (CID) tandem mass spectrometry was performed at a normalized collision energy setting of, typically, approximately 15 % of 5 V, with helium as collision gas, activation q setting of 0.25 and an activation time of 30 ms. Unless otherwise stated, the IT-MS measurements were performed on solutions containing 10 mg Sb L−1 in 50 % methanol or acetonitrile. Triple-quadrupole MS (QqQ-MS) experiments were performed with a Thermo Electron TSQ Quantum AM equipped with an ES interface. ES ionization was achieved by use of 3.7 kV spray voltage with nitrogen as sheath and
The chromatographic column used for separation of Sb–biomolecule complexes was a BDS Hypersil C18 reversed-phase column (2.1 mm×150 mm) from Thermo Electron. Several mobile phases were tested for separation of the complexes on the C18 column but only results obtained using 1 mmol L−1 ammonium formate (pH 6.8–6.9) with 10 % methanol (flow-rate 0.2 mL min−1) used with both ICP–MS and ES–MS detection are presented. The chromatographic column used in the work involving RNA and DNA was a sizeexclusion HEMA-BIO Linear 10-μm (300 mm×8.0 mm) column and the mobile phase was 10 mmol L−1 ammonium acetate (pH 6.8–6.9). For analysis by ES– MS, 10 % methanol was added to the mobile phase.
Results and discussion Characterization of Sb–biomolecule complexes by direct-infusion ES–MS–MS Direct infusion ES–MS analyses of diluted reaction mixtures containing inorganic Sb(V) and one of the
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Sb–biomolecule complexes with 1:1 stoichiometry were initially only observed for reaction of Sb(V) with adenosine, AMP, 3′,5′-CAMP and 2′-deoxyadenosine. Sodium adducts of the Sb-AMP complexes were observed as a result of using the AMP disodium reagent. It was, however, subsequently observed that detection of the 1:1 complexes was very dependent on the nature of the infusion solvent used for electrospray analysis. Performing infusion in a mixture of 1 mmol L−1 ammonium formate and 10 % methanol revealed the general presence of 1:1 complexes, in contrast with their non-detection when using 50 % methanol. The Sb-containing ions observed after each reaction are summarized in Table 1. It is important to note that the response factors for these complexes under ES conditions are not known; it is, therefore, not possible to correlate the observed intensities with the relative amounts of the individual complexes. Adduct ions of the Sb–biomolecule complexes with water, potassium (originating from the potassium hexahydroxoantimonate used as the Sb source), and methanol (the
biomolecules adenosine, cytidine, guanosine, uridine, 2′deoxyadenosine, AMP, 3′,5′-CAMP and ribose (Fig. 1a) afforded mass spectra in which high-mass antimonycontaining ions, other than those corresponding to inorganic Sb(V), were observed. Antimony-containing ions are conveniently detected as a result of the element’s characteristic isotope pattern (121Sb and 123Sb with natural abundances of 57.4 % and 42.6 %, respectively [20]). Ion peaks corresponding to Sb–biomolecule complexes with 1:2 stoichiometry were present after reaction of inorganic Sb(V) with the 8 biomolecules mentioned above. These ions were tentatively assigned as having the general formula: [Sb+2X−3H+OH]+ (suggested structures are presented in Fig. 1b), where X represents the neutral biomolecule. Complexes of Sb(V) with the nucleosides: adenosine, guanosine, cytidine, uridine and 2′-deoxyadenosine of 1:3 stoichiometry were also observed (Table 1). The general formula: [Sb+3X−4H]+ was tentatively assigned to these complexes (Fig. 1b). Fig. 1 Structures of: (a) the biomolecules investigated in this study for their reactivity towards inorganic Sb(V); (b) the observed Sb(X)n (X is a neutral biomolecule; n=1–3) complex ions: Sb(X), complex observed as [Sb+X−H+3OH]+; Sb(X)2, complex observed as [Sb+2X− 3H+OH]+; and Sb(X)3, complex observed as [Sb+3X−4H]+ (arrows indicate possible alternative binding sites for inorganic Sb(V)); c) protonated neutral Sb–biomolecules
a
N
N
N
N
N H
N
N
HN
N
N
H2N
HO O
N
O
O HO
HO HO
OH
Adenosine (Mr = 267)
OH
HO
HO
OH
HO
OH
Ribose (Mr = 150)
N
N
O HO P O HO
O
OH
HO
HO HO
b
O HO O
R
HO
O
HO O HO + Sb OH HO
HO
HO O
O
O
OH
+H+ O
+H+ O
R
R HO
HO HO
HO
O O O Sb OH O
O OH
Sb+
Sb(X)3
R
OH
HO
R O
O
HO
Sb
O O
Sb(X)2
O
R
HO
HO
O O O Sb O O
R
R O
OH Sb(X)2
O R
O
OH
Adenosine-3',5'-cyclic monophosphate (Mr = 329)
O
R
OH
R
+H
Sb(X)
OH
O Sb+ OH O
+
O
O O P O HO
R
Sb(X)
c
O
Adenosine-5'- monophosphate (Mr = 347)
2'-deoxyadenosine (Mr = 251)
N
N
N O
HO
HO
2'-deoxyribose (Mr = 134)
N
N
N
N
N
N
N
O
NH2
NH2
NH2
OH
Uridine (Mr = 244)
Cytidine (Mr = 243)
Guanosine (Mr = 283)
N O
OH
HO
HO
HN
O
O
HO
HO
O
N
N
N
O
Adenine (Mr = 135)
NH2
O
NH2
NH2
OH Sb(X) 3
825 Table 1 Assignments for prominent Sb–biomolecule complex ions and their CID products observed after direct infusion of solutions containing 10 mg Sb L−1 in 50 % methanol and analyzed by ES–MSn in both positive and negative ionization modes Ionization mode
Sb–adenosine +
+
− + − Sb–cytidine +
+
Assignment of precursor ionsb,c
Precursor ion Intensityd
MSn
918–920 783–785 648–650 669–671 534–536 404–406 685–687 480–482 454–456
[Sb+3X−4H]+
1.5×104
[Sb+2X−3H+OH]+
6.2×104
2 3 4 2 3 4
783–785 648–650 518–520 534–536 404–406 136
[P−A]+ [P−A]+ [P−C5H6O4]+ [P−A]+ [P−C5H6O4)]+ [A+H]+
[Sb+2X−3H+OH+O]− [Sb+X−H+3OH+3CH2]+ [Sb+X−H+3OH+O]−
1.5×104 4.4×103(e) 1.8×103
846–848 735–737 624–626 621–623 603–605
[Sb+3X−4H]+
1.4×104
[Sb+2X−3H+OH]+
1.6×104
2 3 4 2 3
735–737 624–626 492–494 603–605 492–494, 510–512 362–364, 381–383
[P−C]+ [P−2C]+ [P−(2C+C5H8O4)]+ [P−H2O]+ [P−(H2O+C)]+, [P–C]+ [P−(H2O+C+C5H6O4)]+, [P–(H2O+2C)]+
966–968 815–817 664–666 532–534 402–404, 381–383 550–552, 152 532–534, 152 402–404, 381–383
[P−H2O]+ [P−(H2O+G)]+ [P−(H2O+2G)]+ [P−(H2O+2G+C5H8O4)]+ [P−(H2O+2G+C10H14O8)]+, [P−(H2O+3G+C5H8O4)]+ [P−G]+, [G+H]+ [P−(G+H2O)]+, [G+H]+ [P−(G+H2O+C5H6O4)]+, [P–(2G+H2O)]+
Precursor ionsa(m/z)
492–494 − Sb–guanosine +
+
Sb–uridine +
+ − Sb–AMP + + − Sb–3′,5′-CAMP +
− + Sb–ribose +
4
Product ionsa (m/z)
Assignment of product ionsf
637–639
[Sb+2X−3H+OH+O]−
2.7×102
984–986 966–968 815–817 664–666 532–534
[Sb+3X−4H+H2O]+
1.2×104
2 3 4 5 6
701–703 550–552 532–534
[Sb+2X−3H+OH]+
1.2×104
2 3 4
849–851 737–739 625–627 623–625 511–513 639–641
[Sb+3X−4H]+
3.2×103
[Sb+2X−3H+OH]+
7.0×103
2 3 4 2 3
737–739 625–627 513–515 511–513 493–495
[Sb+2X−3H+OH+O]−
[P−U]+ [P−2U]+ [P−3U]+ [P−U]+ [P−(U+H2O)]+
1.3×104
829–831 811–813 562–564 516–518
[Sb+2X−3H+OH]+
3.5×103
2 3
811–813 564–566
[P−H2O]+ [P−(H2O+A+CH5O4P)]+
[Sb+X−3H+3OH+2Na]+ [Sb+X−3H+3OH]−
1.4×103
2
500–502
[P−O]−
811–813
[Sb+2X−2H+2OH]+
1.7×104
2
793–795 845–847 500-502
3
793–795, 775–777 641–643
[Sb+2X−2H+4OH]− [Sb+X−H+3OH]+
[P−H2O]+, [P−2H2O]+, [P−(H2O+A+OH)]+
1.5×103 7.4×102
[Sb+2X−4H]+
8.2×103
2 3
399–401 381–383
[P−H2O]+ [P−2H2O]+
417–419 399–401
(e)
826 Table 1 (continued) Ionization mode
− Sb–2′-deoxyadenosine + +
+ Sb–2′-deoxyribose
Precursor ionsa(m/z) 381–383 319–321
Assignment of precursor ionsb,c
[Sb+X−3H+3OH]
−
Precursor ion Intensityd
2.0×10
MSn
Product ionsa (m/z)
Assignment of product ionsf
4
251–253
[P−(2H2O+C5H6O4)]+
2 3 4
502–504 388–390 136
[P−A]+ [P−(A+C5H6O3)]+ [A+H]+
2
908–910 637–639 502–504 388–390 422–424
[Sb+3X−5H+K]+ [Sb+2X−3H+OH]+
1.8×103(e) 2.3×104
[Sb+X−H+3OH]+
1.0×103(e)
–
–
–
–
–
–
–
–
–
–
–
–
Sb–adenine a
Precursor ion corresponding to both Sb isotopes X is a neutral biomolecule See Fig. 1b for structural assignment of precursor ions d ES–MS optimized on the precursor ion e ES–MS not optimized on the precursor ion f P represents precursor ion: A, adenine; C, cytosine; G, guanine; U, uracil b c
presence of solvent adducts was confirmed by testing different solvents), at a variety of intensities, were always observed. Results comparable with those obtained in positiveionization mode were also obtained in negative-ionization mode. More specifically, the 1:1 complexes Sb–adenosine, Sb–AMP, and Sb–ribose, and the 1:2 complexes Sb–adenosine, Sb–cytidine, Sb–uridine, and Sb–(3′,5′CAMP) were detected in the negative ionization mode (the ions observed are summarized in Table 1). The only two biomolecules investigated in this study that did not give any evidence of complexation with inorganic Sb(V), in either negative or positive-ionization mode, were 2′-deoxyribose and adenine. The adenine standard was the only nucleobase tested for complexation with Sb(V). Evidence suggesting lack of complexation of inorganic Sb(V) with the other nucleobases was, however, obtained indirectly from the ES mass spectra of nucleoside solutions, found to contain free nucleobases, to which inorganic Sb(V) had been added. These findings suggest that the interaction between Sb(V) and any nucleobase is insignificant, in agreement with the suggestion of Demicheli et al. [18] that the nucleobase is not involved in the binding of inorganic Sb. Chai et al. [19] reported, on the basis of NMR evidence, “little binding of Sb(V) to the nitrogen (N7) in guanosine”. To characterize the Sb–biomolecule complexes further it was necessary to use multistage MSn. More specifically, identification of the Sb binding site(s) was of paramount interest. Collision-induced dissociation (CID) tandem mass spectrometry of ions corresponding to the 1:2 and 1:3 complexes was performed with an ion-trap multistage MS. These experiments revealed that a common feature of the
CID behavior of the Sb(V)-nucleoside complexes is initial loss of a nucleobase (Table 1). That these covalent bonds dissociate before any of the Sb–biomolecule bonds suggests the high stability of Sb bonding. As shown in Table 1, one, two, or three nucleobases are lost sequentially from most of the complexes. An example of this behavior is shown in Fig. 2 for the 1:3 Sb-guanosine complex. From these experiments it is obvious that the antimony is primarily bound to the ribose or deoxyribose moiety of the biomolecule. Such binding has previously been suggested, but not confirmed, on the basis of NMR measurements [17–19]. The MS and multistage CID results suggest that either one or two of the biomolecule hydroxyl groups are involved in the binding of Sb (Table 1). It is suggested that the 1:2 and 1:3 Sb-ribonucleoside complexes involve binding of Sb to two hydroxyl groups, most probably at the 2′ and 3′-hydroxyl positions (Fig. 1b). Involvement of the 2′ and 3′-hydroxyl positions in the formation of the 1:2 Sb-ribonucleoside complexes is in agreement with earlier observations made by Kluefers and Mayer [17] and Chai et al. [19], but in contrast with the suggested complexation proposed by Demicheli et al. [18], who proposed that only the 2′-ol position participated in the binding. The observed 1:2 complexation between Sb(V) and 3′,5′-CAMP, as indicated by [Sb+2X−2H+2OH]+ (m/z 811, 813), suggests the involvement of a single hydroxyl group in the binding of Sb (in this instance the 2′-ol position or the OH of the phosphate group). The CID results cannot be used to eliminate the possibility of either the 2′ OH group or the OH of the phosphate group participating in the binding. The formation of the 1:2 Sb-2′-deoxyadenosine complex suggested involvement of two hydroxyl groups, in which
827
[Sb(guanosine)3+H2O]+ [Sb(guanosine)2]+
100
[Sb(guanosine)3]+
701
50
719
739
815
984
Full scan
966
850 873 909
0 71
966 968
50
Ms2 983-987@12%
Relative Abundance
815 0 53
815 817
Ms3 965-969@15% 20
-G
0 19
966
664 666
10
Ms4 814-818@15%
O
HO
-G 0 12
5
O
O+ Sb O
815
532 664
O
Ms5 663-667@15%
534 666
OH
O
-C5H8O4
0 4
532
-G 381
404
Ms6 531-535@19%
534
402
2
-C5H6O4
0 400
500
600
700
800
900
1000
m/z
Fig. 2 Representative full MS and MSn spectra of the Sb(guanosine)3 complex formed on reaction of inorganic Sb(V) with guanosine at a ratio of 1:2. G represents the nucleobase guanine. The product ion observed at m/z 381 is noted because it is a common
product ion of several of the observed Sb(biomolecule)n complexes on CID in an ion-trap MS (% values specify collision energy of 5 V maximum used to achieve each CID)
case the 5′-ol position and the 3′-ol position participate in the binding (as indicated by the alternative binding sites shown in Fig. 1b). Assignment of the ions’ positive charge to the Sb atom is supported by the fact that nucleobase product ions are not the main ions observed on CID, because they are lost as neutrals. Only for the adenine and guanine-containing nucleosides were low-intensity nucleobase ions observed. This strongly suggests that the nucleobases are not the major charge-bearing moieties of the complex, i.e. they are not highly protonated under the conditions used in this study, even though they contain basic groups. This is in contrast with the CID behavior observed for the nucleosides adenosine, cytidine, guanosine, and uridine present in control solutions (no antimony added), for which intense nucleobase products ions were observed. The possibility of the existence of protonated neutral Sb–biomolecule complexes cannot be excluded, however (Fig. 1c). Overall, direct infusion ES–MS and MS–MS proved useful for rapid identification and characterization of Sb–biomolecule complexes. Detection of the Sb(adenosine)2 complex could be achieved in reaction mixtures containing a total of 0.5 mg Sb L−1 (full-scan mode; m/z 400–800). One of the primary reasons for choosing MS techniques was their high sensitivity and selectivity, which may enable the development of methods suitable for identification of Sb–biomolecule complexes in biological
samples. These features are an important advantage over other molecule-specific methods, for example NMR spectroscopy (which normally requires major sample clean-up and pre-concentration steps), when used for the detection of analytes present at relatively low concentrations in complex sample matrices. One of the limitations of ES–MS is the difficulty of correlating the relative intensities observed for each of the complexes with the actual amounts present. This is because responses, i.e. ionization efficiency, for each complex may differ substantially, as a result of their strong dependence on the ES source conditions and the different physicochemical properties of the compounds. Extraction of quantitative information can be further complicated by the presence of adducts of potassium and sodium. Characterization of Sb–biomolecule complexes by LC-MS When performing analysis by LC-MS, Sb complexes present in solution can be separated by the LC and detected by MS. Analysis of the inorganic Sb(V) and biomolecule reaction mixtures on a reversed-phase column coupled online with either ICP–MS or ES–MS confirmed the presence, in solution, of 1:1 and 1:2 complexes between Sb(V) and each of the biomolecules adenosine, cytidine,
828
guanosine, uridine, ribose, and AMP (Table 2). Unreacted inorganic Sb(V) was observed to elute at the front of the chromatograms (retention time 1.5 min) for all the reaction mixtures analyzed. Clusters in the m/z range 400–500, 560–660, 730–800, and 900–1000 with a complex isotopic pattern were characteristic of inorganic Sb(V) when analyzed using ES–MS in positive-ion mode. An example of the applied chromatography, with both ICP–MS and ES–MS detection, is shown in Fig. 3. In this instance inorganic Sb(V), Sb(adenosine), and Sb(adenosine)2 species were all well resolved. It is interesting to note that the relative responses to the Sb(adenosine) and Sb(adenosine)2 complexes, i.e. the ratio of the two species, were the same for both detection systems. Because equimolar response of both species is expected in ICP–MS, these data reveal equimolar ES–MS response under the conditions applied. Hence, the ES–MS response may, in this instance, be used to compare the relative amounts of the Sb–adenosine complexes. The Sb(adenosine)2/Sb(adenosine) ratio in this study was found to be between 0.2 and 0.5, indicating that the 1:1 complex was the main species present in solution
(in agreement with Ref. [19]). Assuming equimolar ES–MS response is obtained for the 1:1 and 1:2 complexes of Sb(V) with all biomolecules in this study, the [1:2]/[1:1] ratios are between 0.1 and 3.1, indicating that either the 1:1 or 1:2 complex may be the major species in solution, depending on the identity of the biomolecule. To confirm the equimolar ES–MS response for all biomolecules, however, it is essential to develop an HPLC–ICP–MS method with baseline separation. It should be noted that other Sb(biomolecule)2 complexes were occasionally observed to elute from the reversed-phase column (Table 2). The different retention times observed for these Sb(biomolecule)2 species may be because they occur as isomers in which Sb(V) is attached to different hydroxyl groups (Fig. 1b). Some comments, both general and specific, about the use of HPLC combined with ICP–MS and ES–MS for characterization of inorganic Sb(V) and biomolecule reaction mixtures follow. First, with ICP–MS detection co-eluting Sb species cannot be resolved. For these, ES–MS detection was invaluable for revealing the over-
Table 2 Chromatographic retention times (tR, min) of antimony-containing compounds tentatively identified by ICP–MS (5–10 μg Sb L−1) and by ES–MS (10 mg Sb L−1) in either full-scan mode [m/z 250–1000] or by precursor-ion scanning for product ion m/z 251–253 at 40 V (this isotopic pair is a common fragment ion observed for several Sb–biomolecule complexes) HPLC–ICP–MS tR Adenosine
Cytidine
Guanosine Uridine
Ribose AMP
Sb(V) 3,5-CAMP 2′-Deoxyribose 2′-Deoxyadenosine Adenine a
1.8 – 3.4 8.5 1.4 – – 2.5 1.7 2.2 1.4 1.6 – – 3.5 1.5 – 1.3 – – 2.6b 3.8b 1.5 1.5 1.5 1.5 1.5
HPLC–ES–MS tR 1.5 2.1 3.7 8.0 1.4 1.5 1.7 2.0 2.3 1.7 2.1 1.4 1.5 1.8 2.0 3.8 1.5 1.9 1.3 1.4 1.5 – – 1.5 1.5 1.5 1.5 1.5
Species identity based on full-scan mass spectra Low intensity
b
Full scana Assignment Inorg. Sb(V) Sb(X) Sb(X)2 Sb(X)2+Sb(X)3 Inorg. Sb(V) Sb(X) Sb(X)2 Sb(X)2 Inorg. Sb(V)+Sb(X)3 Inorg. Sb(V) Sb(X)+Sb(X)2 Inorg. Sb(V) Sb(X)2 Sb(X)2 Sb(X)2 Sb(X)2 Inorg. Sb(V)+Sb(X) Sb(X)2 Sb(X)2 Sb(X) Inorg. Sb(V) – – Inorg. Sb(V) Inorg. Sb(V) Inorg. Sb(V) Inorg. Sb(V) Inorg. Sb(V)
HPLC–ES–MS tR – 2.1 3.4 – – – 1.7 2.0 2.2 – 2.2 – 1.5 1.8 2.0 3.4 – 1.9 – – – – – – – – – –
Precursor-ion scan of m/z 251–253 Assignment – Sb(X) Sb(X)2 – – – Sb(X)2 Sb(X)2 Sb(X)2 – Sb(X)2 – Sb(X) Sb(X)2 Sb(X)2 Sb(X)2+Sb(X)3 – Sb(X)2+Sb(X)3 – – – – – – – – – –
829
a
b
+
[Sb(adenosine)] 438
Peak 2
300000
440
6000000 452 454
200000 390
c
410
430
451
471
491
511
531
551
571
591
611
631
651
Sb
4000000
368
2+
[Sb(adenosine)2]
121
346
Peak 1 Sb(V)
Intensity
Intensity QqQ-MS
Fig. 3 (a) Reversed phase HPLC chromatograms obtained from Sb–adenosine complexes by use of ICP–MS detection (dotted line) and ES–MS detection for the m/z ranges 437.5– 440.5 and 668.5–671.5 (solid line); (b) mass spectrum of peak 2 corresponding to Sb(adenosine); and (c) the mass spectrum of peak 3 corresponding to Sb (adenosine)2
335 +
[Sb(adenosine)2]
336
669 671
100000 2000000
251
305
355
406
456
507
559
611
669
719
770
820
m/z
Peak 3 0
0 0
1
2
3
4
5
6
7
8
9
10
11
12
Retention time (min)
lapping species (Table 2). Second, not all Sb–biomolecule species detected in direct infusion ES–MS were observed when using HPLC–ES–MS and HPLC–ICP–MS. More specifically, only the presence of 1:3 complexes between Sb(V) and adenosine or cytidine were observed by HPLC– ES–MS in full-scan mode (Table 2). For both of these signal intensity was low and the failure to detect other 1:3 complexes may be partly because of the smaller amounts of reaction mixture analyzed with the latter two techniques. Also, in contrast with direct infusion ES–MS, neither HPLC–ES–MS nor HPLC–ICP–MS analysis provided evidence for complexation between inorganic Sb(V) and 2′-deoxyadenosine or 3′,5′-CAMP. It is possible these complexes are formed in the gas phase during direct infusion, whereas for HPLC analysis only complexes preexisting in solution can be observed. The Sb content of both reaction mixtures eluted quantitatively from the column in the void volume together with inorganic Sb (HPLC–ICP–MS), hence failure to detect the Sb species by HPLC–ES–MS is not because Sb complexes are retained on the column, i.e. these complexes either decompose during chromatography or do not exist in solution. None of the three techniques used in this study provided evidence for complexation of inorganic Sb(V) with 2′-deoxyribose or adenine. The Sb content of these two reaction mixtures also eluted quantitatively from the HPLC column in the void volume together with inorganic Sb(V). The failure to detect any complexes between Sb and 2′-deoxyadenosine, 3′,5′-CAMP, 2′-deoxyribose, or adenine when using HPLC strengthens the view that vicinal hydroxyl groups are critical for their formation in solution.
Studying the complexation of Sb(V) with RNA and DNA oligomers To test this hypothesis, i.e. that vicinal hydroxyl groups are essential for formation of the Sb–biomolecule complexes in question, an RNA and an DNA oligomer were also tested for complexation with Sb(V). Because of the general instability of short RNA compared with DNA, the analyzed RNA oligomer was longer than the DNA oligomer (15 base pairs compared with 5). Solutions of Sb(V), DNA, RNA, and 1:1 mixtures of Sb and DNA and of Sb and RNA were analyzed by size-exclusion HPLC–ICP–MS. Sb(V) eluted as one peak after 8.7 min and the retention times of DNA and RNA were determined, by monitoring 31P, to be at 8.0 and 7.5 min, respectively. Complexation of Sb(V) with the RNA oligomer was observed, but not complexation with the DNA oligomer (Fig. 4). The yield of the Sb–RNA complex increased at lower pH. At pH 7, 3.7±0.2 % total Sb was determined to form an RNA complex, compared with 5.6±1.3 % at pH 5. Applying heat during incubation (1 h at 37 °C, pH 5) further increased the amount of complex formed (11.1±1.2 % of total Sb). Increasing the molar ratio of Sb(V) relative to RNA also increased the total amount of Sb–RNA complex. The Sb present in all the analyzed samples eluted quantitatively from the column. Because no positive results were obtained from attempts to complex Sb(V) with DNA, only the results for RNA are discussed further. A mixture of the RNA oligomer with potassium carbonate (potassium carbonate was added to furnish a concentration of potassium similar to that in the
830
modes. In the positive-ionization mode the triply charged RNA oligomer (m/z 1585) was mainly observed and no RNA–Sb complex was detectable. In the negativeionization mode, however, there were some differences between the mass spectrum of the RNA oligomer and that of the RNA–Sb(V) mixture (Fig. 5). The RNA oligomer was detected mainly with charges of −4 (m/z 1187.5) and −5 (m/z 949.8) (several adducts of water and K are present). The mass spectrum of the RNA–Sb(V) mixture contained additional small peaks at m/z 1258.5 and m/z 1007.0 (again several adducts of water and K are present). This suggests the presence of RNA–Sb(V) complexes of 1:1 stoichiometry which contain three potassium ions and carry the charges of −4 and −5, respectively (Fig. 5). These data are, to the best of our knowledge, the first direct evidence of the ability of Sb(V) to form a complex with RNA. It proved difficult to obtain good mass spectra from the samples, possibly because of interference from the potassium ions, and it was not possible to obtain MS–MS results for the Sb–RNA complex to verify its identity. To improve the MS data (and to overcome the interference from K) prospective studies may include analysis of the Sb(V)–RNA complex by HPLC–ES–MS–MS. It can be expected that binding of Sb to vicinal diols may affect the general stability and function of RNA and the subject deserves more attention. Fig. 4 Size exclusion HPLC chromatograms obtained from (a) a mixture of the DNA oligomer and Sb(V) (showing no binding) and (b) a mixture of the RNA oligomer and Sb(V) (ca 10 % binding) detected by ICP–MS (31P shown in grey and 121Sb in black). A HEMA-BIO chromatographic column was used and the mobile phase was 10 mmol L−1 ammonium acetate (1 mL min−1)
sample in which inorganic Sb(V) was added), RNA–Sb mixture, and DEPC-treated water (blank) were analyzed by direct-infusion NS–MS in positive and negative-ionization Fig. 5 Mass spectra of (a) a solution of RNA and potassium (1:5) and (b) a mixture of RNA and Sb (1:5) measured by directinfusion NSI–ES–MS in negative ionization mode. Asterisks indicate m/z from the proposed Sb(V)–RNA complex
Effect of reaction conditions on Sb–biomolecule formation Attempts were made to investigate the effect of changing reaction conditions, for example pH, reaction time, and Sbto-biomolecule molar ratio, on the extent of Sb–biomolecule formation. On the basis of the ES–MS results it was obvious that some complexation between inorganic Sb(V)
831
Development of tandem mass spectrometric methods for detecting trace levels of Sb–biomolecule complexes Selected reaction monitoring (SRM) and precursor-ion scanning were the two tandem MS approaches investigated for their suitability for detection of trace levels of Sb–biomolecule complexes. To develop these approaches it was necessary to construct CID breakdown curves for the detected Sb(biomolecule)n (n=1–3) complexes over the
a
100 90 80
Sb(V) in reaction (%)
70 < 10 min 6h 24 h
60 50 40 30 20 10 0
2
3
4
5
6
7
8
9
10
11
Reaction pH
b
90 80
Relative Amount (%)
70 Sb(adenosine)
60 50 40
Sb(V)
30 20 Sb(adenosine)2
10 0 0
4
8
12
16
20
24
215
Reaction Time (h)
c 1,4x10
4
1,2x10
4
1,0x10
4
8,0x10
3
6,0x10
3
4,0x10
3
2,0x10
3
1,0x10
Sb(adenosine)
6
8,0x10
Sb(adenosine)3
6
6,0x10
Sb(adenosine)2
6
4,0x10
6
2,0x10
0,0
Intensity of Sb(adenosine)3
7
Intensity of Sb(adenosine)n (n =1, 2)
and the biomolecules occurred immediately after mixing at room temperature. Because the Sb–biomolecule species distribution observed by direct-infusion ES–MS depended on conditions such as the carrier solvent used for ES, the ES source settings, and the individual compound ionization efficiency, it was necessary to analyze the reaction mixtures by HPLC–ICP–MS and HPLC–ES–MS also. When using HPLC–ICP–MS to analyze the reaction mixture of Sb(V) with adenosine it was observed that complexation occurred at pH 3, 5, and 7, but not to a detectable extent at pH 10 (Fig. 6a). Complex formation was favored at pH<7. Demicheli et al. [18], who used less sensitive NMR and CD techniques, also reported complex formation at pH 5 but not at neutral pH. That the reaction also occurs at pH 7 indicates the reaction may be of greater physiological importance than previously thought. Reaction time was found to have a significant effect on the amount of Sb(adenosine) and Sb(adenosine)2 formed, as shown in Fig. 6b. The 1:1 complex was formed more quickly than the 2:1 complex, in agreement with recent findings by Chai et al. [19], who found that formation of the 1:1 complex of Sb(V) with GMP was more than ten times faster than that of the 2:1 complex. From Fig. 6b it is apparent that maximum formation of the Sb(adenosine) complex occurred after 6 h; this was followed by slow formation of Sb(adenosine)2. This observation supports the suggestion made by Chai et al. [19] that the reaction between Sb(V) and GMP or GDP-mannose occurs in two steps. When the reaction mixtures were stored at 4 °C the relative amounts of unreacted inorganic Sb(V) remained rather constant for 9 days (35±0.6 %, n=3) but the relative amount of Sb(adenosine) decreased by 15 % and the amount of Sb(adenosine)2 increased correspondingly (data not shown). Increasing the molar reaction ratio of adenosine-to-Sb resulted in an increase of the intensities of the peaks for all three Sb–adenosine complexes, and the 1:3 complex, in particular, was favored at higher ratios (Fig. 6c). Heating the Sb–adenosine reaction (1:2 molar ratio) for 2 h at 37 °C increased the yields of both the 1:1 and 1:2 complexes, but none of the 1:3 complex was observed (Fig. 6c). Increasing both the molar ratio and the storage time increased the number of different Sb(adenosine)n species present. In contrast with direct infusion ES–MS, results from both HPLC–ICP–MS and HPLC– ES–MS showed that the 1:1 Sb–adenosine complex was the most dominant species in solution for all reaction conditions tested.
0,0
0
2
4
6
8
10
Molar ratio adenosine/Sb
Fig. 6 The effect of reaction conditions on Sb–adenosine complex formation was studied for reaction mixtures containing 100 mg Sb L−1 by HPLC–ICP–MS (0.5 mg Sb L−1) and by HPLC–ES–MS (10 mg Sb L−1): (a) variation of the relative amount of inorganic Sb (V), determined by HPLC–ICP–MS, in the reaction mixture (% of total eluted Sb) is shown as a function of reaction pH; (b) the relative distribution (% of total eluted Sb) of inorganic Sb(V), Sb (adenosine), and Sb(adenosine)2 as a function of reaction time (reaction pH 5) determined by HPLC–ICP–MS; (c) the intensity of Sb(V), Sb(adenosine), and Sb(adenosine)2, determined by HPLC– ES–MS, in reactions performed at pH 5 with different adenosine-toSb(V) molar ratios. The encircled symbols represent values for reactions heated for 2 h at 50 °C. All values are averages from 2 or 3 measurements; standard deviations were negligible and are thus not shown
832 5000
Sb(adenosine)2 tR = 3.7
3000 2000 1000 0 0
1
2
3
4
5
Retention time (min)
Fig. 7 Reversed phase SRM ES–MS–MS chromatogram obtained from a liver homogenate extract (dotted line) and an extract of the liver homogenate spiked with the Sb(V)–adenosine reaction mixture to give a total concentration of 15 mg Sb L−1 (solid line). These signals correspond to the sum of the SRM transitions monitored, i.e. m/z 668.5–671.5→m/z 533.5–536.5 at 25 eV and m/z 668.5– 671.5→m/z 250.5–253.5 at 50 eV
collision energy range from 10–60 eV (CID breakdown curves for most of the Sb–biomolecule complexes are provided as Electronic Supplementary Material Figs. S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, and S13). These breakdown curves revealed precursor ion–product ion transitions suitable for use in SRM, and the presence of product ions common to most of the Sb–biomolecule complexes examined in this study, and thus potentially suitable for use in precursor-ion scanning. The SRM conditions used for detection of the Sb (adenosine)2 complex were: m/z 668.5–671.5→m/z 533.5– 536.5 at 10 eV (this transition represents loss of an adenine nucleobase) and m/z 668.5–671.5→m/z 250.5–253.5 at 30 eV. When using this SRM method in combination with reversed phase HPLC, a detection limit below 25 μg Sb L−1 (total Sb concentration in the reaction) was obtained for the Sb(adenosine)2 complex. To evaluate this approach further a portion of the Sb(V)–adenosine reaction mixture was spiked into a crude liver homogenate (tissue/water=1:4) to give a total concentration of 15 mg Sb L−1. Analysis by reversed-
2 1.2E+05 Intensity of a and b
Fig. 8 Reversed-phase chromatograms of the Sb–adenosine reaction mixture detected by ES– MS using: (a) precursor-ion scanning (m/z 250.5–253.5 at 40 eV); (b) SRM (m/z 668.5– 671.5→m/z 533.5–536.5 at 10 eV and m/z 668.5–671.5→m/ z 250.5–253.5 at 30 eV); and (c) full-scan mode (m/z [668.5– 671.5] extracted). Peak 1 corresponds to Sb(adenosine) and peak 2 to Sb(adenosine)2. Peak 3 gives a mass spectrum identical with that of Sb(adenosine)2. Ion peaks marked with asterisks were not found to contain Sb
1.2E+06
2
9.0E+04
9.0E+05 *
6.0E+04
1
2
*
3 3
3.0E+04
6.0E+05
c b
3.0E+05
a 0.0E+00
0.0E+00 0
2
4
6
8
Retention time (min)
10
12
14
Intensity of c
Intensity
4000
phase HPLC, with SRM ES–MS–MS, of a sample taken from this mixture revealed the presence of a low-intensity Sb(adenosine)2 peak with an elution time of 3.7 min, despite the extremely complex nature of the analyzed sample and the possibility of competing complexation and enzymatic activity having occurred (Fig. 7). Assuming no matrix effects occurred during HPLC–ES–MS–MS analysis it was estimated that less than 5 % of the spiked complex remained in the liver homogenate at the time of analysis. To reduce the possibility of this being an artifact, a blank liver homogenate and spiked samples containing higher concentrations of Sb– adenosine were also analyzed. No peak corresponding to Sb (adenosine)2 was detected in the blank liver homogenate, and samples spiked with higher concentrations of Sb confirmed the retention time of the 1:2 Sb–adenosine complex. No further attempts were made to identify all the other peaks observed in both the control and spiked liver homogenate extracts. Although SRM is ideal for targeted analysis it lacks the ability to detect unknown compounds. To compensate for this the precursor ion-approach was explored by using common product ions observed for several of the Sb– biomolecule complexes studied here (m/z 251 and 253, and m/z 381 and 383). The precursor ion method developed on the basis of the m/z 251 and 253 product ions was capable of detecting most of the Sb(X) and Sb(X)2 compounds present in the reaction mixtures. A summary of the species detected by use of the precursor-ion scan of 251–253 (m/z 250.5–253.5) is presented in Table 2. This approach is, however, less sensitive than full-scan mode or the SRM mode, so not all expected precursor ions were detected. Chromatograms obtained from the Sb–adenosine reaction mixture recorded in different MS scanning modes are compared in Fig. 8. In this instance precursor-ion scanning, SRM, and full-scan mode were used. The intensity obtained in the full-scan mode is 10 times higher than that by any other mode, but the background noise is also much higher. Also, the lack of selectivity in the full-scan mode results in chromatographic peaks which do not contain Sb.
833
Conclusions ES–MS–MS is useful for rapid identification and characterization of Sb–biomolecule complexes formed in vitro at relatively low concentrations (10 mg Sb L−1). Sb–biomolecule complexes with a stoichiometry of 1:1, 1:2, or 1:3 were detected and characterized by directinfusion ES–MS–MS after reaction of inorganic Sb(V) with each of the studied biomolecules except 2′-deoxyribose and adenine. Definitive evidence for the formation of Sb-complexes in solution was provided for complexes of Sb(V) with all the analyzed biomolecules with vicinal cis hydroxyl groups (adenosine, cytidine, guanosine, uridine, and AMP by use of both HPLC–ICP–MS and HPLC–ES–MS, for ribose only by use of HPLC– ES–MS, and for an RNA oligomer only by use of HPLC–ICP–MS). The physiological reaction conditions under which inorganic Sb(V) forms relatively stable complexes with biomolecules containing vicinal cis hydroxyl groups suggests that such complexes may occur in vivo. It was shown that the Sb(adenosine)2 complex was sufficiently stable to be detected one hour after being spiked into a liver homogenate (the total concentration of the spike was 30 mg Sb L−1) despite competing complexation and any enzymatic activity which might have occurred. SRM with ES–MS–MS in combination with HPLC enabled successful detection of the spiked Sb complex (actual concentration unknown) in the liver homogenate, despite the extremely complex nature of the analyzed sample. Combination of MS methods, as used in this study, has potential for future use in the identification of such complexes in biological samples. Acknowledgements The authors thank the European Commission for the funding of a Marie Curie Excellence Grant (Project name: ACE-METALS, Contract No. MEXT-CT-2003-002788) and Dr Mina Tsagri (Institute of Molecular Biology and Biotechnology, Crete, Greece) for donating the DEPC used in the work with RNA/ DNA and for her valuable advice on the handling of RNA.
References 1. Ouellette M, Drummelsmith J, Papadopoulou B (2004) Leishmaniasis: drugs in the clinic, resistance and new developments. Drug Resist Updat 7:257–266 2. Winship KA (1987) Toxicity of antimony and its compounds. Adv Drug React Ac Pois Rev 2:67–90 3. Andrewes P, Kitchin K, Wallace K (2004) Plasmid DNA damage caused by stibine and trimethylstibine. Toxicol Appl Pharmacol 194:41–48
4. International Agency for Research on Cancer (1989) Monographs on the evaluation of carcinogenic risk to humans: some organic solvents, resin monomers and related compounds, pigments and occupational exposures in paint manufacture and painting, vol 47. IARC Lyon, French 5. Zheng J, Iijima A, Furuta N (2001) Complexation effect of antimony compounds with citric acid and its application to the speciation of antimony(iii) and antimony(v) using HPLC–ICP– MS. J Anal At Spectrom 16:812–818 6. Lintschinger J, Koch I, Serves S, Feldmann J, Cullen WR (1997) Determination of antimony species with high-performance liquid chromatography using element specific detection. Fresenius J Anal Chem 359:484–491 7. Yan S, Li F, Ding K, Sun H (2000) Complexation of Antimony (III) by trypanothione. Angew Chem Int Ed 39:4260–4262 8. Sun H, Cheong S, Cheng WS (2000) Interaction of antimony tartrate with the tripeptide glutathione. Implication for its mode of action. Eur J Biochem 267:5450–5457 9. Roberts WI, McMurray WJ, Rainey PM (1998) Characterization of the antimonial antileishmanial agent meglumine antimonate (glucantime). Antimicrob Agents Chemother 42:1076–1082 10. Dodd M, Pergantis SA, Cullen WR, Hao L, Eigendorf GK, Reimer KJ (1996) Antimony speciation in freshwater plant extracts by using hydride generation-gas chromatography-mass spectrometry. Analyst 121:223–228 11. Andrewes P, Cullen WR, Feldmann J, Koch I, Polishchuk E (1999) Methylantimony compound formation in the medium of Scopulariopsis brevicaulis cultures: 13CD3-L-methionine as a source of the methyl group. Appl Organometal Chem 13: 681–687 12. Feldmann J, Koch I, Cullen WR (1998) Complementary use of capillary gas chromatography-mass spectrometry (ion trap) and gas chromatography-inductively coupled plasma mass spectrometry for the speciation of volatile antimony, tin and bismuth compounds in landfill and fermentation gases. Analyst 123: 815–820 13. Feldmann J, Hirner AV (1995) Occurrence of volatile metal and metalloid species in landfill and sewage gases. Int J Environ Anal Chem 60:339–359 14. Lintschinger J, Schramel O, Kettrup A (1998) The analysis of antimony species by using ESI–MS and HPLC–ICP–MS. Fresenius J Anal Chem 361:96–102 15. Pezzano H, Podo F (1980) Structure of binary complexes of mono- and polynucleotides with metal ions of the first transition group. Chem Rev 80:365–401 16. Ralston NVC, Hunt CD (2001) Diadenosine phosphates and S-adenosylmethionine: novel boron binding biomolecules detected by capillary electrophoresis. Biochim Biophys Acta 1527:20–30 17. Kluefers P, Mayer P (1997) Polyol-metall-komplexe. 27. Bisdiolato antimonates(III) with guanosine as the diol. Z Anorg Allg Chem 623:1496–1498 18. Demicheli C, Frezard F, Lecouvey M, Garnier-Suillerot A (2002) Antimony(V) complex formation with adenine nucleosides in aqueous solution. Biochim Biophys Acta 1570: 192–198 19. Chai Y, Yan SC, Wong ILK, Chow LMC, Sun HZ (2005) Complexation of antimony (Sb-V) with guanosine 5′-monophosphate and guanosine 5′-diphospho-D-mannose: formation of both mono- and bis-adducts. J Inorg Biochem 99:2257–2263 20. Handbook of Chemistry and Physics (1990) CRC Press, Boca Raton, FL, 71st edition
