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 first- line treatment drug against Leishmania parasites. Using HPLC-inductively coupled plasma-MS and electrospray- MS, 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 com- plexes 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 mol- ecules 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 depro- tonated 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- 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 chromato- graphic 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 * To whom correspondence should be addressed. Tel.: +45 3533 6283. Fax: +45 3533 6010. E-mail: hrh@farma.ku.dk. † 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. (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. Anal. Chem. 2008, 80, 5993–6000 10.1021/ac800677u CCC: $40.75  2008 American Chemical Society 5993 Analytical Chemistry, Vol. 80, No. 15, August 1, 2008 Published on Web 06/14/2008