Characterization of Sodium Stibogluconate by ... - ACS Publications

Jun 14, 2008 - Glaxo Operations UK Limited (Bernard Castle, UK). Throughout the study ..... Peak 1 was mainly composed of the 1:1 Sb-gluconate complex...
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Anal. Chem. 2008, 80, 5993–6000

Characterization of Sodium Stibogluconate by Online Liquid Separation Cell Technology Monitored by ICPMS and ESMS and Computational Chemistry Helle Ru¨sz Hansen,*,† Claus Hansen,† Kasper P. Jensen,‡ Steen Honore ´ Hansen,† Stefan Stu¨rup,† † and Bente Gammelgaard Department of Pharmaceutics and Analytical Chemistry, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen Ø, Denmark, and Department of Chemistry, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark High-performance liquid chromatography (HPLC), mass spectrometry (MS), and computational chemistry has been applied to resolve the composition and structure of the Sb species present in dilutions of Pentostam, a firstline treatment drug against Leishmania parasites. Using HPLC-inductively coupled plasma-MS and electrosprayMS, it was shown that the original drug consists of large Sb(V)-glyconate complexes of polymeric nature that degrade upon dilution. In dilution solution, the drug is a mixture of noncomplexed Sb(V), large polymeric complexes as well as several low molecular mass Sb(V)glyconate complexes of various stoichiometry (1:1, 1:2, 1:3, 2:2, 2:3, 2:4, 3:3, 3:4). The 1:1 complex became the most abundant low molecular mass Sb(V) complex with dilution time. A novel mixed-mode chromatographic system was applied in order to separate complexes of various stoichiometry and isomers. Density functional theory was used to study the structure of the 1:1 Sb-gluconate complex with three or four solvent molecules bound. By computing the structures and the free energies of the various possible isomers in aqueous solvation models, the most likely structures of the species were deduced. Importantly, 6-coordination is always preferred over 5-coordination, and the species commonly adopt conformations involving tris-coordination of deprotonated hydroxyl groups from gluconate. Although pentavalent antimony(Sb(V))-containing drugs have been used for treatment of millions of patients suffering from leishmaniasis since the 1930s, little is known about the chemistry and biochemistry of these drugs.1–4 This may be partly due to the fact that the disease is most predominant in less developed areas such as Africa, Asia, and Latin America. However, restric* To whom correspondence should be addressed. Tel.: +45 3533 6283. Fax: +45 3533 6010. E-mail: [email protected]. † University of Copenhagen. ‡ Technical University of Denmark. (1) The World Health Report 1996; World Health Organization: Geneva, 1996. (2) Yan, S.; Jin, L.; Sun, H. In Metallotherapeutic Drugs and Metal-Based Diagnostic Agents. The Use of Metals in Medicine, 1st ed.; Gielen, M., Tiekink, E. R. T., Eds.; John Wiley & Sons: Hoboken, NJ, 2005; Chapter 23. (3) Berman, J. D. Rev. Infect. Dise. 1988, 10, 560–86. 10.1021/ac800677u CCC: $40.75  2008 American Chemical Society Published on Web 06/14/2008

tions in the analytical accessibility constitute another reason. So far, advances within the area of antimony speciation have been limited, compared to advances in speciation of other metalloids such as arsenic and selenium.5 Understanding the detailed chemical structure of a drug is essential in order to establish its mechanism of efficiency and to device further improvements in drug development, with reduced side effects. Progress within the area of metal or metalloid speciation has been tremendous during the past decade, mainly due to the rapid development of coupled analytical techniques.6 Especially the coupling of high-performance liquid chromatography (HPLC) to inductively coupled plasma- (ICP) and electrospray- (ES) mass spectrometry (MS) has been successful.6 However, for antimony species, stability and chromatographic elution/separation have been problematic. Thus, only a very few studies have reported successful antimony speciation using chromatographic separation. One main limitation has been the tendency of Sb(III) species to stick to column materials, necessitating the use of chelating mobile phases for its quantitative elution.7 For this reason, nonvolatile antimony speciation studies have been mainly restricted to the separation of antimony in its two oxidation states (Sb(V)/Sb(III)),7 where information about the original ligands is lost. In a critical study of available thermodynamic data for the complexation of antimony(III) and antimony(V) with organic ligands of low molecular mass, no data could be found for Sb(V) complexes and only a few for Sb(III) complexes.8 Although recent chromatographic approaches have resulted in the identification of several low molecular mass Sb(V) complexes,9–14 there is a need for research into alternative separation systems, in order to deduce the biological relevant forms of Sb complexes. Presently, Sb(V) complexed to polyhydroxy carbohydrates in the form of sodium stibogluconate or meglumine antimonate (4) Frezard, F.; Martins, P. S.; Barbosa, M. C. M.; Pimenta, A. M. C.; Ferreira, W. A.; deMelo, J. E.; Mangrum, J. B.; Demicheli, C. J. Inorg. Biochem. In press. (5) Hansen, H. R.; Pergantis, S. A. Inductively Coupled Plasma Spectrometry and its Applications, 2nd ed.; Hill, S. J., Ed.; Blackwell Publishing: Ames, IA, 2006; Chapter 8. (6) Lobinski, R.; Schaumloffel, D.; Szpunar, J. Mass Spectrom.Rev. 2006, 25, 255–89. (7) Krachler, M.; Emons, H.; Zheng, J. Trends Anal.Chem. 2001, 20, 79–90. (8) Filella, M.; May, P. M. J. Environ. Monit. 2005, 7, 1226–37.

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(Trade names: Pentostam and Glucantime, respectively) are used as the agents of first choice in the treatment of the parasitic disease leishmaniasis.15 Despite the common use of Pentostam or Glucantime, their chemical structures are not well defined2,4 and extremely high doses are used in treatment (typically intravenously at a dosage of 20 Sb mg kg-1 day-1 for 30 days).3,16 These high doses often cause serious side effects in the patients (showing signs of metal toxicity), as well as development of resistant parasites.15 A recent study reported on “new insights into the structure and composition of meglumine antimonate and sodium stibogluconate” by use of direct infusion ESMS and osmolarity measurements.4 The study confirmed earlier results for meglumin antimonate, which were obtained by direct infusion fast atom bombardment-MS, ESMS and by nuclear magnetic resonance spectroscopy.17,18 More specifically, it was seen that meglumin antimonate consists of a mixture of oliogomeric structures of the general formula (NMG-Sb)-NMG and (NMG-Sb)n (where NMG is N-methyl-D-glucamin) and that the main species detectable by ESMS were 1:1 and 1:2 Sb-NMG complexes. Direct infusion ESMS measurements of sodium stibogluconate also showed that it consists of a mixture of oligomeric structures, in agreement with earlier results obtained by molecular sieve chromatography,19 and consistent with the general formula for meglumin antimonate ((SSG-Sb)n-SSG and (SSG-Sb)n (SSG, sodium stibogluconate)). Osmolarity measurements suggested the predominance of the 1:1 Sb-NMG and Sb-SSG complexes in diluted samples.4 Furthermore, structures of the main stibogluconate complexes (Sb-SSG; 1:1 (Figure 1), 1:2, 2:2, and 2:3) were proposed, which involved a preferred coordination of Sb(V) to 4,5-O of gluconic acid and linkage of Sb atoms by gluconate where applicable. The most often quoted structure, a 2:2 complex, involves coordination of Sb(V) to 2,4-O20 and 2,3,4-O2,19,21,22 and linkage of Sb atoms by an oxygen. Although the structure is commonly quoted, its analytical origin is unclear and thus it is unknown under which conditions the structure has been deduced. It is possible that it may have deferred from a crystal structure, with neutral total charge, whereas Pentostam is a solution. Thus, the understanding of the chemical structure of this drug has not reached any consensus, and the need for alternative approaches for structure elucidation is warranted. (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22)

Hansen, H. R.; Pergantis, S. A. J. Anal. At. Spectrom. 2006, 21, 1240–48. Hansen, H. R.; Pergantis, S. A. J. Anal. At. Spectrom. 2006, 21, 731–3. Hansen, H. R.; Pergantis, S. A. Anal. Bioanal. Chem. 2006, 385, 821–33. Guy, A.; Jones, P.; Hill, S. J. Analyst 1998, 123, 1513–8. Chai, Y.; Yan, S. C.; Wong, I. L. K.; Chow, L. M. C.; Sun, H. Z. J. Inorg. Biochem. 2005, 99, 2257–63. Hansen, H. R.; Pergantis, S. A. Anal. Chem. 2007, 79, 5304–11. Croft, S. L.; Sundar, S.; Fairlamb, A. H. Clin. Microbiol. Rev. 2006, 19, 111–26. Markle, W. H.; Makhoul, K. Am. Fam. Physician 2004, 69, 1455–60. Roberts, W. L.; McMurray, W. J.; Rainey, P. M. Antimicrob. Agents Chemother. 1998, 42, 1076–82. Demicheli, C.; Ochoa, R.; Lula, I. S.; Gozzo, F. C.; Eberlin, M. N.; Frezard, F. Appl. Organomet. Chem. 2003, 17, 226–31. Berman, J. D.; Grogl, M. Exp. Parasitol. 1988, 67, 96–103. St.George, S.; Bishop, J. V.; Titus, R. G.; Selitrennikoff, C. P. Antimicrob. Agents Chemother. 2006, 50, 474–9. Beveridge, E. In Experimental Chemotherapy; Schnitzer, R. J., Hawking, F., Eds.; Academic Press: New York, 1963; Chapter 6. Findlay, G. M. Recent advances in chemotherapy, 3rd ed.; Findlay, G. M., Ed.; J & A Chruchill Ltd.: London, 1950; Chapter 6.

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Figure 1. Proposed structural formula for the 365, 367 Da ions given by Frezard et al.4

As pointed out by Frezard et al.,4 direct infusion ESMS spectra do not accurately reflect the composition of the starting material, since detection efficiency is species specific and typically decreases with increasing mass. Also, certain electrically neutral or zwitterionic species cannot be identified by this technique and species may form as artifacts in the electrospray source. Thus, data obtained by direct infusion ESMS have to be evaluated with great care, and the data can in general not be used for drawing any quantitative conclusions. The main objectives of this study were to obtain quantitative speciation data on possible, biologically relevant Sb(V) species in Pentostam and to suggest accurate structures of these using complementary computational chemistry. Although Sb(III) is believed to be the active form of Sb in Sb-containing drugs,2,23 the structure of the pentavalent antimony drug will be crucial for the distribution and uptake of Sb initially by the macrophage and then by the parasite. We apply a novel mixed-mode chromatographic system, which together with state-of-the-art computations provides a powerful approach to structure elucidation of physiologically relevant Sb complexes. EXPERIMENTAL SECTION Materials and Reagents. Potassium hexahydroxoantimonate (Sb(V)) was acquired from Fluka (Buchs, Switzerland); ammonium formate (>98%), sodium chloride, and sodium hydroxide were from Riedel-de Hae¨n (Seelze, Germany); citric acid monohydrat was from Merck (Darmstadt, Germany); hydrochloric acid (30%) was from VWR International (Albertslund, Denmark); methanol and acetonitrile were from BDH Chemicals (Lutterworth and Poole, England, respectively), D-gluconic acid was from Sigma (St. Louis, MO), formic acid (96%) was from Aldrich (Steinheim, Germany), and D-glucose was from Nordisk Droge (Copenhagen, Denmark). The Pentostam drug, Sodium Stibogluconate Injection BP, had a Sb concentration of 100 mg/mL and was acquired from Glaxo Operations UK Limited (Bernard Castle, UK). Throughout the study, Millipore deionized water (18 MΩ cm) from a Milli-Q Plus Ultrapure water system from Millipore (Bedford, MA) was used. All reagents were of analytical grade. (23) Miekeley, N.; Mortari, S. R.; Schubach, A. O. Anal. Bioanal. Chem. 2002, 372, 495–502.

Sb(V) stock solution (12.0 mmol/L) was prepared by dissolving potassium hexahydroxoantimonate in water. A standard of Sb(V)-gluconate complexes was produced by mixing Sb(V) (0.82 mmol/L) with a solution of D-gluconic acid (8.2 mmol/L). The solutions of Sb(V) and Pentostam were typically diluted to contain 10 mg/L Sb (for ICPMS analysis) and 100 mg/L (for analysis by ESMS) with either water, isotonic saline (0.9%), glucose (5%), or blood plasma as specified in the text. Analytical Information. An Agilent 1100 series HPLC pump system (Agilent Technologies, Palo Alto, CA) equipped with an Agilent 1200 series microinjector was used. The analytical size exclusion chromatography (SEC) column was a Shodex GS-320 (7.6 × 300 mm), and the flow rate was 0.6 mL/min mobile phase consisting of a mixture of 50 mmol/L ammonium formate and 10% methanol. The mixed-mode analytical column was an Obelisc N (2.1 × 150 mm, 5 µm) from SIELC. In the optimized conditions, the mobile phase consisted of a mixture of 36 mmol/L ammonium formate and 10% acetonitrile (pH 4.0), and the flow rate was 0.1 mL/min. The injection volume was 0.1 and 1 µL for analysis by ICPMS and ESMS, respectively. The ICPMS instrument was a PE-Sciex ELAN 6000 (PerkinElmer, Norwalk, CT) equipped with a cyclonic spray chamber (Glass Expansion) and a pneumatic nebulizer. PEEK tubing was used to connect the outlet of the HPLC column directly to the inlet of the ICP nebulizer. Signals at m/z 121 and 123 (corresponding to 121Sb and 123Sb) were measured. Data analysis was performed on a TotalChrom workstation (Perkin-Elmer). HPLC-ESMS measurements were performed on a HewlettPackard series 1100 MSD single quadropole mass spectrometer (Hewlett-Packard, Palo Alto, CA) with an electrospray ionization interface operating in negative ionization mode. Nitrogen was used as nebulizing gas at a pressure of 40 psi at 12 L/min, the temperature was 350 °C, and the capillary voltage was 4000 V. A piece of PEEK tubing was used for connecting the outlet of the LC directly to the ESMS. The instrument was operated at variable cone voltage (specified in text) in the full scan mode or in the selected ion monitoring mode (SIM). In the SIM method, a cone voltage of 120 V was applied and the following m/z values were measured: 223, 225, 365, 367, 543, 545, 695, 697, 699, 703, 705, 819, 821, 823, 953, 955, 957, 1015, 1017, 1019, 1131, 1133, 1135. Computational Chemistry. Theoretical calculations were carried out with the Turbomole program,24 version 5.8. All optimizations were carried out in redundant internal coordinates using the Cosmo model25,26 iteratively during geometry optimization, to yield structures that are minimums on the potential energy surfaces in solution. Based on earlier experience and assessment of accuracy,27 the Becke 1988 exchange functional combined with the Perdew 1986 nonlocal correlation functional (BP86)28,29 were used. The basis sets used for geometry optimization were def2SVP for all atoms.30 The Cosmo calculations were performed with a dielectric constant of 80, similar to water. For the generation of (24) Alrichs, R.; Ba¨r, M.; Ha¨ser, M.; Horn, H.; Ko ¨lmel, C. Chem. Phys. Lett. 1989, 162, 165–9. (25) Klamt, A.; Schu ¨u ¨ rmann, J. J. Chem. Soc., Perkin Trans. 1993, 2, 799–805. (26) Scha¨fer, A.; Klamt, A.; Sattlel, D.; Lohrenz, J. C. W.; Eckert, F. Phys. Chem. Chem. Phys. 2000, 2, 2187–93. (27) Jensen, K. P.; Roos, B. O.; Ryde, U. J. Chem. Phys. 2007, 126, 014103. (28) Becke, A. D. Phys. Rev. A 1988, 38, 3098–100. (29) Perdew, J. P. Phys. Rev. B 1986, 33, 8822–4. (30) Weigend, F.; Alrichs, R. Phys. Chem. Chem. Phys. 2005, 7, 3297–305.

the cavity, a set of atomic radii has to be defined. The optimized Cosmo radii in Turbomole were applied (H, 1.30 Å; O, 1.72 Å; C, 2.00 Å).31 The radii used for Sb were 2.0 Å. Free energies of each isomer were computed using frequency analysis. In this way, the entropy, which can vary substantially between isomers depending on the number of oxides bound from the gluconic acid, is taken into account and contributes to the final free energy of each isomer. Gluconic acid, i.e., (2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanoic acid, was modeled with Sb coordinated by two or three oxide atoms and possibly by the carboxylate. The m/z 365 and 383 fragments have been studied, corresponding to (i) two deprotonated hydroxyl groups coordinating to Sb, and three additional hydroxyl groups bound to Sb (m/z 365), with a charge of Q ) -1; (ii) the protonated isomers of this species, with Q ) 0; (iii) an additional water bound to Sb (m/z 383), with charge Q ) -1; and (iv) the protonated isomers of this species, with Q ) 0. All coordination modes and protonation states of carboxylate and solvent molecules were evaluated, and several conformations of each isomer compared when necessary. RESULTS AND DISCUSSION In order to establish the physiologically relevant species in a drug formulation, it is important to perform the analytical work in accordance with clinical treatment procedures. This is because any physiological change, such as dilution and pH change, will change the equilibrium between Sb(V) and ligands. Unfortunately, no exact guidelines exist on how to perform the intravenous injection of Pentostam, apart from that it should be administered very slowly over 5 min to reduce the risk of local thrombosis (accordingly to the Pentostam product description). To ease the administration, Pentostam may be diluted with 5% dextrose (2.5-4 times)16 or, according to the procedure at the Danish Rigshospital, by slow infusion with isotonic saline (treatment over 2-3 h). Thus, various “dilution procedures” exist, so in this study, both dilution with blood plasma, isotonic saline and glucose (5%) were investigated. The infusion procedure may potentially be important for the treatment efficiency, due to impacts on the equilibrium. Analysis by Size Exclusion LC. As Pentostam tends to form oligomers, an initial approach is the use of SEC coupled with ICPMS and ESMS. The SEC-ICPMS measurements of diluted Pentostam samples revealed that the distribution of Sb species in the formulation changed as a function of dilution time from high molecular weight complexes toward low molecular weight complexes and that the degradation rate increased with temperature (Figure 2). The drug formulation was subsequently analyzed by SECESMS in both the negative and positive ionization modes. The negative ionization mode was found most sensitive, and several complexes of Sb and gluconic acid were detected, which can all be described by the general formula [Sbm(Glu(OH)6)n(OH)p (5m - p + 1)H]- (Table 1). Apart from the m/z values reported for complexes of different stoichiometry in Table 1, both more and less hydrated species were observed (m/z ±18). When applying a low cone voltage, detection of the more hydrated species was generally favored (e.g., m/z 695/697/699 compared (31) Klamt, A.; Jonas, V.; Bu ¨ rger, T.; Lohrenz, J. C. W. J. Phys. Chem. A 1998, 102, 5074–85.

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Figure 2. Chromatograms from SEC of Pentostam diluted with plasma and analyzed by ICPMS (m/z 121) after storage for (a) 1 h at 5 °C, (b) 24 h at 5 °C, (c) 24 h at 20 °C, and (d) 48 h at 37 °C (gray). Table 1. Assignment of Prominent Ions Observed in Pentostam Dilutions Analyzed by SEC-ESMS and LiSC-ESMS (last column)

m/z of main ions 365/367 543/545 695/697/699 703/705 819/821/823 953/955/957 1015/1017/1019 1131/1133/1135 general formula

interpretation [Sb(Glu(OH)6)(OH)3 - 3H][Sb(Glu(OH)6)2(OH)2 - 4H][Sb2(Glu(OH)6)2(OH)4 - 7H][Sb2(Glu(OH)6)3 - 6H][Sb2(Glu(OH)6)3- 11H][Sb3(Glu(OH)6)3(OH) - 15H][Sb2(Glu(OH)6)4 - 11H][Sb3(Glu(OH)6)4 - 16H][Sbm(Glu(OH)6)n(OH)p (5m - p + 1)H]-

peak number in Figure Sb:Glu 3, 4 1:1 1:2 2:2 1:3 2:3 3:3 2:4 3:4

1 2 3 4, 6, 7 8,9

to m/z 641/643/645). Thus, the ESMS data cannot reveal the actual number of binding sites between Sb(V) and gluconate in solution as the results depend on the ES source settings. The fragmentation pattern of Pentostam was further investigated by use of ESMS/MS, but as no valuable structural information could be obtained (due to gluconate and the Sb(V) complexes’ ability to defragment water), these data are not reported here. As the complexes of different stoichiometry showed slightly different retention times, it is unlikely that the complexes formed as artifacts in the ES source. Most of the observed complexes fitted the general formulas produced by Frezard et al.4 ((SSG-Sb)n-SSG and (SSG-Sb)n), except the complexes where the number of gluconate was more than one unit higher than of Sb (Sb-gluconate; 1:3 and 2:4). The 3:4 Sb-gluconate complex was the largest detectable; however, as larger complexes were generally harder to be ionized (requires a higher cone voltage), it is possible that even larger complexes were not ionized in the ES source and were thus simply not detectable. Analysis by Liquid Separation Cell (LiSC) Technology. In order to obtain quantitative data on the distribution of the Sb-gluconate complexes in the Pentostam formulation, an improved chromatographic separation to SEC was required. Using reversed-phase (RP) LC, mixed-mode RP/cation-exchange LC, cation-exchange LC, or zwitterionic hydrophilic interaction liquid 5996

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chromatography LC, poor or no separation between species was obtained. Although some separation was observed on LC columns with anion-exchange capacities (LUNA-NH2, Hamilton PRP-X100), the chromatographic recovery was generally very poor and unsatisfactory, unless chelating ligands such as EDTA were added to the mobile phase. As the challenge was to identify the original Sb(V)-ligand complexes in Pentostam, the addition of complexing ligands had to be avoided. Subsequently, a column ideal for charged polar compounds was applied. Obelisc N is one of the first commercially available columns with LiSC. LiSC technology is based on a new chemical modification of silica gel pores that creates a liquid separation cell with its own charge characteristics, ionic strength, and hydrophobic propertiesslike a living cell. It contains both positively and negatively charged groups and thus has the ability of interacting with both positively or negatively charged or zwitterionic species. At least seven Sb peaks were always observed in the LiSCICPMS chromatograms when diluted with water, isotonic saline (0.9%), or glucose (5%). The relative distribution of Sb species did not differ between the dilution media. Peaks 1-3 and 6-9 were always present in the chromatograms (Figure 3). Peak 5 only occurred when a sample was measured immediately after dilution. Peak 4 coeluted with the tailing of peak 3 and was thus not always visible in the HPLC-ICPMS chromatograms. When noncomplexed Sb(V) was incubated with excess of D-gluconic acid (1:10), three Sb peaks coeluting with peaks 1-3 in Pentostam were observed (spike shown in Figure 3 inset). This suggests that the Sb species giving rise to peaks 1-3 in Pentostam dilutions have relatively simple stoichiometries, based on comparison with results obtained on similar incubations of Sb(V) with lactate14 and citrate9 (1:1, 1:2, 1:3). The influence of mobile-phase pH and composition on the retention times of Sb peaks was investigated. Decreasing the ion strength or increasing the relative amounts of organics led to slower elution of the Sb peaks but did not improve the resolution. The retention times were markedly influenced by pH, and increasing pH to the highest recommended (4.5) led to shorter retention times but poor resolution. At this pH, a standard of noncomplexed Sb(V) (potassium hexahydroxyantimonate) eluted as a broad tailed peak with the eluent, but at pH 4.0, it was almost

Figure 3. LiSC-ICPMS chromatograms (m/z 121) of Pentostam when analyzed immediately (gray) and 0.5 h after dilution (black) at room temperature (20 °C) with 0.9% isotonic saline and using the optimized chromatographic conditions. The inset show the 0.5-h dilution (solid) spiked with a mixture of Sb(V) and D-gluconic acid (dotted).

Figure 4. LiSC-SIM-ESMS chromatograms of Pentostam (diluted for 2 h at 20 °C). The m/z values of the colored chromatograms are as follows: (black) 365, 367; (blue) 543, 545; (green) 695, 697, 699; (red) 819, 821, 823; and (gray) 953, 955, 957. Note that the real intensity of peak 1 is 10 times higher than shown.

fully retained on the column. The optimal separation of Sb species in Pentostam was obtained using a mobile phase of 36 mmol/L ammonium formate, 10% acetonitrile, and pH 4.0. Under these conditions, noncomplexed Sb(V) did not elute as a peak, but instead gave rise to an elevated background signal. The Sb species occurring in solutions of Pentostam (Figure 3) were identified by LiSC-SIM-ESMS (Figure 4, Table 1). Only the identity of peak 5 could not be accounted for by the SIM method. Identification of Sb species in Pentostam by full-scan MS was only possible for peak 1 due to low sensitivity. When a solution of gluconic acid was injected as a blank, only one peak occurred, which eluted at 4.2 min (eluent front). Gluconic acid (M ) 196) was detected mainly as m/z 223, possibly due to adduct formation with formate in the mobile phase. As no peak was observed for m/z 225, the m/z 223 peak was not due to [Sb(OH)6]-.

Peak 1 was mainly composed of the 1:1 Sb-gluconate complex, but low intensity signals of Sb-gluconate complexes of higher stoichiometry (1:2, 1:3, 2:2, 2:3) were also observed (Figure 4). As the peak of noncomplexed gluconic acid was overlapping with peak 1, it may be speculated that the minor signals of Sb-gluconate complexes of higher stoichiometry observed in peak 1 are gasphase artifacts generated in the ES source. Peaks 2 and 3 showed the identity of a 1:2 and 2:2 complex, respectively. Peaks 4, 6, and 7 shared identity, suggesting either the occurrence of isomeric species or the presence of higher stoichiometry Sb-gluconate complexes only detectable as fragments due to absent ionization of the original complex. The presence of isomers seems to be the most reasonable explanation, as the mass spectra showed the presence of the 2:3 Sb-gluconate complexes both when low and high cone voltage was applied and no other fragments were Analytical Chemistry, Vol. 80, No. 15, August 1, 2008

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Figure 5. Relative distribution (as percentage of total Sb) of Sb peak 1, the 1:1 Sb(V)-gluconate complex (squares), peak 3, the 2:2 Sb(V)-gluconate complex (triangles), and chromatographic recovery (circles) in Pentostam when analyzed by LiSC-ICPMS (Figure 3), depicted as a function of time (h) from dilution of sample at room temperature (20 °C).

observed in the relevant peaks, as would be expected upon fragmentation. The signals of peaks 8 and 9 were very weak, but also indicated the occurrence of isomers (3:3 Sb-gluconate complexes). The occurrence of isomers for Sb(V)-lactate complexes has previously been suggested.14 Although m/z 953, 955, and 957 all show a small peak at the retention time of peak 5, their relative distribution does not match the isotopic pattern of the 3:3 Sb(V)-gluconate complex. The fact that several isomers of each Sb(V)-gluconate stoichiometry can occur severely complicates the Sb speciation study. Nevertheless, it is shown for the first time that complexes of Sb(V) with gluconate of various stoichiometries, 1:1, 1:2, 1:3, 2:2, 2:3, 2:4, 3:3, and 3:4, may exist in solution. On the basis of osmolarity measurements, Frezard et al.4 have previously suggested the predominance of the 1:1 Sb(V)-gluconate complex in Pentostam dilutions. In the present work, we found that the 1:1 and 2:2 Sb-gluconate complexes coexist in equal quantities in fresh dilutions of the drug and that, with increasing dilution time, the 1:1 complex became more dominant (Figure 5). These observations suggest that the 1:1 Sb complex is a prominent species in serum of patients. The 1:2 complex was always less significant, in contrast to observations by direct infusion ESMS.4 This implies the importance of applying a quantitative method such as HPLC-ICPMS in combination with an identification technique. With increasing dilution time. both the dominance of the 1:1 complex and the chromatographic Sb recovery increased (defined as percent eluted Sb of total Sb) (Figure 5). This suggests the degradation of larger complexes (which did not elute of the column) to the 1:1 complex. It should be noted that this experiment was performed at 20 °C and that the degradation rate at physiological temperature is expected to be higher (in accordance with Figure 2). In the analyzed diluted solution (10 mg/ L), an apparent equilibrium was established after 1.5 h (Figure 5). However, both degradation of larger complexes to the 1:1 complex (increasing chromatographic recovery) and degradation of the 1:1 complex to noncomplexed Sb(V) (decreasing chromatographic recovery) may occur within this time frame, possibly explaining why the chromatographic recovery never exceeded a 5998

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maximum of 50%. Addition of citric acid, a known chelate of Sb(V), increased the chromatographic recovery of Sb in the Pentostam dilution (from 45 to 65% in a 1.5-h-old dilution), as a large peak of Sb(V)-citrate eluted with a retention time of 5 min. This suggests the presence of noncomplexed Sb(V) in the Pentostam dilutions, as the distribution of other peaks did not change. Addition of HCl to the Pentostam dilutions also increased the chromatographic Sb recovery, mainly due to a large increase in the intensity of peak 1, suggesting the hydrolysis of larger complexes into the 1:1 Sb-gluconate complex. Thus, the 50% of antimony species not accounted for may constitute a mixture of noncomplexed Sb(V) and large polymeric Sb-gluconate complexes retained on the column. Although major advances in chromatographic separation of Sb species and major insights into the complexity of antimony’s chemistry have been presented in this study, it is obvious that some limitations, such as incomplete chromatographic recovery, exist. Also, although HPLC-ICPMS and HPLC-ESMS has been successfully applied in identifying different stoichiometries of Sb(V)-gluconate complexes present in solution, the nature of the isomers has not been revealed, and the use of complementary methods was deemed necessary. Computational Chemistry. Computational chemistry was applied in order to deduce the most likely structure of the 1:1 Sb(V)-gluconate complex. Density functional theory is in principle capable of describing accurately chemical systems and is thus of invaluable use in the search for the biologically active isomers, all this assuming that a physically correct functional is to be applied.32 It is known that the choice of functional critically affects many properties of chemical systems outside the range for which the density functional are originally parametrized. In contrast, geometries of complexes are generally quite well modeled by any state-of-the-art functional, with errors in computed geometries expected to be ∼0.03 Å for bond lengths of simple transition metal complexes;27 similar errors can be expected for the Sb complexes. Such accuracy allows careful assignment of low-energy isomers of molecular systems, in particular when free (32) Kohn, W.; Becke, A. D.; Parr, R. G. J. Phys. Chem. 1996, 100, 12974–80.

Figure 6. Most stable isomer of (a) M366 (detected by ESMS at m/z 365, 367) with Q ) -1:5A and (b) M384 (detected by ESMS at m/z 383, 385) with Q ) -1:2C. The green atom is Sb(V), red atoms are oxygen, black are carbon, and white are hydrogen.

energies are taken into account, and for the first time allows us to deduce the biological relevant forms of Pentostam consistent with experimental data. It should be stressed, in agreement with comments made in the introduction, that the biological relevance of the Sb(V)-gluconate complexes is probably associated with uptake and not actual action on the parasite. The different coordination modes refer to the numbering of the carbons of the main chain of gluconic acid, with additional solvent molecules (3 or 4) bound to Sb: The isomers possible include coordination to Sb in various forms, i.e., O1/O2, O1/O3, O4/O5, O1/O2/O3, and O3/O4/O5 (see Figure 1 for numbering of O). These forms distinguish the use of two and three oxygens of the gluconic acid coordinating to Sb, where three is possible when only three solvent molecules are bound, as the maximum coordination number of Sb is six. The 1:1 complex was detected mainly at m/z 365, 367 and 383, 385 by HPLC-ESMS and will be referred to as M366 and M384, respectively, in the following. Under neutral or weakly acidic conditions, M366 and M384 are most likely negatively charged (Q ) -1), as observed by ESMS, or neutral (Q ) 0). For each of the four combinations, 8-11 isomers were geometry optimized, and the free energies computed based on frequencies and thermodynamic analysis, as described in the Experimental Section. These results are found in Supporting Information (SI). Some species have the same general structure, i.e., stoichiometry, number of bound hydroxyl groups, and types of solvent molecules bound to Sb; however, these isomers still differ in terms of positions of protons and hydrogen bonds, which are not accounted for in the SI, since only the most stable isomers have been discussed. For M366 carrying one negative charge (Q ) -1), the most stable isomer (5A) exhibits an O1/O2/O3 coordination mode (Figure 6a, SI), which is the maximum coordination number when three solvent molecules (in this case, three OH-) are bound. It is 18 kJ/mol more stable than another O1/O2/O3 isomer (11A) and 19 kJ/mol more stable than the O3/O4/O5 isomer (1A), which also has three OH- groups bound to Sb (SI). Although 5A has a charge of -1, it is expected to be fairly basic and easily take up a proton. If that happens, the proton can go in several places, but two forms have particularly low free energy, which is

a O1/O2/O3 complex with three OH- bound to Sb (11B, SI) and a O3/O4/O5 complex with one H2O and two OH- bound to Sb (2B, SI). These structures have the same free energy within 3 kJ/mol and are expected to be in equilibrium in solution. Thus, we can conclude that the three species, 5A (Figure 6a), 11B, and 2B (SI) constitute the peak at m/z 365, 367 depending on the details of the solution (pH): 5A dominates at higher pH, whereas the other forms are found at lower pH. We cannot calculate the pH where this change occurs since we cannot compute absolute pKas of the individual species. All of them imply that three oxygens of the gluconic acid coordinate to Sb, to maximize the coordination number of Sb and thereby maximize charge neutralization. Again, we cannot calculate the pH where this change occurs, but can rule out a variety of other structural forms, including the simple form proposed for the 365 Da ion (Figure 1) by Frezard et al.4 For M384, one extra H2O molecule has been bound to the complex, forcing one of the hydroxyl groups of the gluconic acid to be displaced by water. When Q ) -1, the most stable isomer is clearly O4/O5 coordination to gluconic acid with 3OH- and H2O also bound to Sb (2C, Figure 6b). It is 19 kJ/mol more stable than the O1/O2 species with four OH- bound to Sb (10C, SI), where the missing solvent proton is now located on a hydroxyl group not coordinating to Sb, and 28 kJ/mol more stable than the O1/O3 species (5C, SI). When M384 is protonated (Q ) 0), it forms several low-energy species, consisting of O1/O2 (6D and 7D) and O4/O5 (1D) coordination, with either 3 OH- and H2O bound to Sb (O1/O2) or with one extra proton to form H2O (O4/ O5) from the extra coordinating alkoxide group of gluconic acid (SI). We can thus conclude that the peak at m/z 383, 385 consists of an O4/O5 coordinated species at higher pH and a mixture of O1/O2 and O4/O5 species at lower pH. In particular, we have shown that in aqueous solution, 6-coordination is always preferred over 5-coordination suggested earlier,2,19,21,22 even for the m/z 365, 367 species where only three solvent molecules are present, forcing three alkoxide oxygens of gluconic acid to coordinate to Sb. Oxide forms of solvent molecules bound to Sb are generally 72-145 kJ/mol less stable than hydroxide and water forms and will not be found in solution. Analytical Chemistry, Vol. 80, No. 15, August 1, 2008

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CONCLUSION The present work represents a combined strategy of using chromatography, mass spectroscopy and computational chemistry to elucidate the main composition and structure of physiologically relevant Sb(V) species found in the drug Pentostam. It has been found that (i) the drug in solution is a mixture of 1:1, 1:2, 1:3, 2:2, 2:3, 2:4, 3:3, and 3:4 stoichiometries (referring to Sb:gluconate) as well as noncomplexed Sb(V) and large polymeric Sb-gluconate complexes; (ii) among the low molecular mass Sb(V) complexes, the 1:1 form dominates in solution, in particular over time; and (iii) several 1:1 structural isomers have been deduced as present in solution, depicted in Figure 6. These complexes are very likely candidates for the physiologically relevant forms of Pentostam. ACKNOWLEDGMENT The authors thank Professor Ib Bygbjerg, Department of International Health, Institute of Public Health, University of

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Copenhagen, and Dr. Joergen Kurtzhals and Dr. Ming Chen, Department of Clinical Microbiology, Rigshospitalet, Copenhagen, for valuable discussions on the subject and donation of Pentostam. K.P.J. acknowledges the Velux foundation for funding and the Danish Center for Scientific Computing (DCSC) for a computing grant.

SUPPORTING INFORMATION AVAILABLE Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs. org.

Received for review April 4, 2008. Accepted May 19, 2008. AC800677U