New method for the speciation of ruthenium-based chemotherapeutics in human serum by conjoint liquid chromatography on affinity and anion-exchange monolithic disks
- Author(s)
- Anze Martincic, Radmila Milacic, Janja Vidmar, Iztok Turel, Bernhard K. Keppler, Janez Scancar
- Abstract
An important step in pharmacological characterisation of a candidate drug is the study of the drugs interactions with serum proteins. In the present work, conjoint liquid chromatography (CLC) was used for separation of ruthenium (Ru)-based drug candidates in human serum. CIM Protein G and CIM DEAE disks were assembled together in a single housing forming a CLC monolithic column. By applying isocratic elution with Tris-HCl-NaHCO
3 buffer (pH 7.4) in the first min, followed by gradient elution with 1molL
-1 NH
4Cl (pH 7.4) in the next 9min, immunoglobulins (IgG) were retained by the Protein G disk enabling subsequent separation of unbound Ru species from Ru species bound to human serum transferrin (Tf) and albumin (HSA) on the CIM DEAE disk. Finally, elution with acetic acid (AcOH) in the next 3min allowed separation of Ru species associated with IgG. Protein elution was followed on-line with UV detection at 278nm, while the separated Ru species were quantified by post-column isotope dilution inductively coupled plasma mass spectrometry (ID-ICP-MS). The instrumental set-up enabled fast two-dimensional separation by affinity and ion-exchange modes to be carried out in a single chromatographic run. Two Ru-based chemotherapeutics: a newly synthesised compound chlorido(η
6-p-cymene)(nalidixicato-κ
2O,O)Ru(II) (1) and (H
2im)[trans-Ru(III)Cl
4(Him)
2] (2; KP418), which is currently undergoing preclinical studies, were investigated. The CLC procedure applied is sensitive with low limit of detection (LOD) (0.027μgRumL
-1 for (1)) and good method repeatability (RSD±3.5%). The experimental data revealed that it enables investigation of the kinetics of interaction of positively charged and neutral complexes of metallodrugs with serum proteins as well as the distribution of metallodrug species in human serum. However, negatively charged metallic complexes co-eluted with Tf and HSA and thus hindered their speciation analysis. An example of successful application of the kinetic studies on the CLC column is (1), a neutral Ru complex that hydrolyses to a positively charged species. For comparison, speciation data obtained for serum samples spiked with cisplatin are also shown.
- Organisation(s)
- Department of Inorganic Chemistry
- External organisation(s)
- Jožef Stefan Institute (IJS), University of Ljubljana
- Journal
- Journal of Chromatography A
- Volume
- 1371
- Pages
- 168-176
- No. of pages
- 9
- ISSN
- 0021-9673
- DOI
- https://doi.org/10.1016/j.chroma.2014.10.054
- Publication date
- 12-2014
- Peer reviewed
- Yes
- Austrian Fields of Science 2012
- 301904 Cancer research, 106023 Molecular biology, 104002 Analytical chemistry
- Keywords
- ASJC Scopus subject areas
- Analytical Chemistry, Biochemistry, Organic Chemistry
- Sustainable Development Goals
- SDG 3 - Good Health and Well-being
- Portal url
- https://ucrisportal.univie.ac.at/en/publications/e113d566-80bc-499f-b0a6-3a3d629760d1