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Development of an LC–MS/MS method for analysis of interconvertible Z/E isomers of the novel anticancer agent, Bp4eT

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Abstract

This study was focused on a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method development for quantification of a novel potential anticancer agent, 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT), in aqueous media. Solid Bp4eT was found to consist predominantly of the Z isomer, while in aqueous media, both isomers coexist. Sufficient separation of both isomers was achieved on a Synergi 4u Polar RP column with a mobile phase composed of 2 mM ammonium formate, acetonitrile, and methanol (30:63:7; v/v/v). The photo diode array analysis of both isomers demonstrated different absorption spectra which hindered UV-based quantification. However, an equal and reproducible response was found for both isomers using an MS detector, which enables the determination of the total content of Bp4eT (i.e., both E− and Z− isomeric forms) by summation of the peak areas of both isomers. 2-Hydroxy-1-naphthylaldehyde 4-methyl-3-thiosemicarbazone (N4mT) was selected as the internal standard. Quantification was performed in selective reaction monitoring using the main fragments of [M+H]+ (240 m/z for Bp4eT and 229 m/z for N4mT). The method was validated over 20–600 ng/ml. This procedure was applied to a preformulation study to determine the proper vehicle for parenteral administration. It was found that Bp4eT was poorly soluble in aqueous media. However, the solubility can be effectively improved using pharmaceutical cosolvents. In fact, a 1:1 mixture of PEG 300/0.14 M saline markedly increased solubility and may be a useful drug formulation for intravenous administration. This investigation further accelerates development of novel anticancer thiosemicarbazones. The described methods will be useful for analogs currently under development and suffering the same analytical issue.

Chromatogram of the LC-MS/MS analysis of Bp4eT; UV absorption spectra of both Bp4eT isomers.

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Acknowledgements

This study was supported by the grant of Ministry of Education, Youth, and Sport of the Czech Republic MSM 0021620822. D.R.R. and D.S.K. thank the National Health and Medical Research Council for grant and fellowship support.

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Authors and Affiliations

  1. Department of Pharmaceutical Chemistry and Drug Control, Faculty of Pharmacy in Hradec Králové, Charles University in Prague, Heyrovského 1203, 500 05, Hradec Králové, Czech Republic

    Ján Stariat, Petra Kovaříková & Jiří Klimeš

  2. Iron Metabolism and Chelation Program, Department of Pathology and Bosch Institute, Blackburn Building, University of Sydney, Sydney, New South Wales, 2006, Australia

    Danuta S. Kalinowski & Des R. Richardson

Authors
  1. Ján Stariat
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  2. Petra Kovaříková
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  3. Jiří Klimeš
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  4. Danuta S. Kalinowski
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  5. Des R. Richardson
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Correspondence to Petra Kovaříková or Des R. Richardson.

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Stariat, J., Kovaříková, P., Klimeš, J. et al. Development of an LC–MS/MS method for analysis of interconvertible Z/E isomers of the novel anticancer agent, Bp4eT. Anal Bioanal Chem 397, 161–171 (2010). https://doi.org/10.1007/s00216-009-3448-7

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  • DOI: https://doi.org/10.1007/s00216-009-3448-7

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